Basal HIF-1a expression levels are not predictive for radiosensitivity of human cancer cell lines

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Schilling, D.; Multhoff, G.; Helmholtz Center Munich, CCG - Innate Immunity in Tumor Biology, Munich; Bayer, C.; Emmerich, K.; Molls, M.; Vaupel, P.; Huber, R.M.

2012-01-01

High levels of hypoxia inducible factor (HIF)-1a in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1a on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1a levels. HIF-1a levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1a, HIF-2a, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1a siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. According to their basal HIF-1a status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1a expressors. The functionality of the high basal HIF-1a expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1a. Linear regression analysis revealed no correlation between basal HIF-1a levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. Our data suggest that basal HIF-1a levels in human tumor cell lines do not predict their radiosensitivity under normoxia. (orig.)

Hydrophilic CeO2 nanocubes protect pancreatic β-cell line INS-1 from H2O2-induced oxidative stress

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Lyu, Guang-Ming; Wang, Yan-Jie; Huang, Xue; Zhang, Huai-Yuan; Sun, Ling-Dong; Liu, Yan-Jun; Yan, Chun-Hua

2016-04-01

Oxidative stress plays a key role in the occurrence and development of diabetes. With their unique redox properties, CeO2 nanoparticles (nanoceria) exhibit promising potential for the treatment of diabetes resulting from oxidative stress. Here, we develop a novel preparation of hydrophilic CeO2 nanocubes (NCs) with two different sizes (5 nm and 25 nm) via an acetate assisted hydrothermal method. Dynamic light scattering, zeta potential measurements and thermogravimetric analyses were utilized to investigate the changes in the physico-chemical characteristics of CeO2 NCs when exposed to in vitro cell culture conditions. CCK-8 assays revealed that the CeO2 NCs did not impair cell proliferation in the pancreatic β-cell line INS-1 at the highest dose of 200 μg mL-1 over the time scale of 72 h, while being able to protect INS-1 cells from H2O2-induced cytotoxicity even after protein adsorption. It is also noteworthy that nanoceria with a smaller hydrodynamic radius exhibit stronger antioxidant and anti-apoptotic effects, which is consistent with their H2O2 quenching capability in biological systems. These findings suggest that nanoceria can be used as an excellent antioxidant for controlling oxidative stress-induced pancreatic β-cell damage.Oxidative stress plays a key role in the occurrence and development of diabetes. With their unique redox properties, CeO2 nanoparticles (nanoceria) exhibit promising potential for the treatment of diabetes resulting from oxidative stress. Here, we develop a novel preparation of hydrophilic CeO2 nanocubes (NCs) with two different sizes (5 nm and 25 nm) via an acetate assisted hydrothermal method. Dynamic light scattering, zeta potential measurements and thermogravimetric analyses were utilized to investigate the changes in the physico-chemical characteristics of CeO2 NCs when exposed to in vitro cell culture conditions. CCK-8 assays revealed that the CeO2 NCs did not impair cell proliferation in the pancreatic β-cell line INS-1 at

Radiosensitization of C225 on human non-small cell lung cancer cell line H-520

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Zhang Yingdong; Wang Junjie; Liu Feng; Zhao Yong

2008-01-01

Objective: To investigate the efficacy of C225 (cetuximab), a chimeric human-mouse anti-epithelial growth factor receptor monoclonal antibody, combined with 60 Co gamma irradiation against human non-small cell lung cancer cell line H-520. Methods: H-520 cells were treated either with different dose of 60 Co irradiation (1,2,4,6,8 and 10 Gy)alone or together with C225 (100 nmol/L). Colony forming capacity was determined to create the survival curve 10 days after the treatment. Cells in different groups were harvested 72 hours after irradiation for apoptosis analysis or 48 hours after irradiation for cell cycle analysis by flow cytometry assay. Results: The clone number in combinational treatment group was less than that in irradiation only group, which suggested that the cell survival rate in the combinational treatment group was significantly decreased comparing with irradiation only group (F=6.36, P O + G 1 phases for C225 treatment, in G 2 + M phases for 60 Co irradiation, and in both G 0 + G 1 and G 2 + M phases for C225 in combination with 60 Co irradiation. Conclusions: C225 has radiosensitizing effects on H-520 cells, which may through the enhancement of 60 Co irradiation-induced cell death and cell cycle arrest. This study provides a supportive evidence for clinical treatment in non-small cell lung cancer. (authors)

Glucocorticoid effect on melphalan cytotoxicity, cell-cycle position, cell size, and [3H]uridine incorporation in one of three human melanoma cell lines

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Benckhuijsen, C.; Osman, A.M.; Hillebrand, M.J.; Smets, L.A.

1987-01-01

Three human melanoma cell lines of known content of specific glucocorticoid-binding sites were studied for colony formation after a microM dose of glucocorticoid combined with melphalan. In one of the three cell lines, M-5A, subcloned from M-5 (formerly designated RPMI 8322), the effect of combined treatment was markedly increased compared to that of melphalan even if the glucocorticoid was applied for 1 h only, 10 h before the melphalan. Semilogarithmic dose-effect plots for a reduction of final plating efficiency by glucocorticoid were curvilinear, according to a receptor-mediated process. The effects of glucocorticoid, melphalan, and their combination were linearized by bilogarithmic median-effect plotting which allowed the quantitation of a synergism which was more marked in case of glucocorticoid pretreatment, for 1 or 24 h, than on simultaneous exposure. According to sequential DNA per cell cytophotometry, melphalan abolished in M-5A a glucocorticoid-induced arrest in the G1 phase of the cell cycle. The cytotoxic synergism correlated with an apparent stimulation by glucocorticoid of the rate of acid-insoluble incorporation of [ 3 H]uridine and [ 14 C]leucine and an increase in cell size and protein content in M-5A cells but not in the other two cell lines. The way in which glucocorticoids induce an enhanced susceptibility to melphalan is not clear. Our results appear compatible with a hypothesis that chromatin in a transcriptionally activated state is more vulnerable to cytotoxic attack by an alkylating agent than under average conditions

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

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Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

2017-10-17

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Bactericidal impact of Ag, ZnO and mixed AgZnO colloidal nanoparticles on H37Rv Mycobacterium tuberculosis phagocytized by THP-1 cell lines.

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Jafari, Alireza; Mosavari, Nader; Movahedzadeh, Farahnaz; Nodooshan, Saeedeh Jafari; Safarkar, Roya; Moro, Rossella; Kamalzadeh, Morteza; Majidpour, Ali; Boustanshenas, Mina; Mosavi, Tahereh

2017-09-01

The purpose of this research project was to infection of human macrophages (THP-1) cell lines by H 37 Rv strain of Mycobacterium tuberculosis (H 37 RvMTB) and find out the ratio/dilution of mixture silver (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) whose ability to eliminate phagocytized bacteria compared to rifampicin. The colloidal Ag NPs and ZnO NPs were synthesized and their characteristics were evaluated. The THP-1 cell lines were infected with different concentration of H 37 RvMTB. Next, the infected cells were treated with different ratios/dilutions of Ag NPs, ZnO NPs and rifampicin. The THP-1 were lysed and were cultured in Lowenstein-Jensen agar medium, for eight weeks. The TEM and AFM images of NPs and H 37 RvMTB were supplied. It is observed that Ag NPs, 2 Ag :8 ZnO and 8 Ag :2 ZnO did not have any anti-tubercular effects on phagocytized H 37 RvMTB. Conversely, ZnO NPs somehow eliminated 18.7 × 10 4  CFU ml -1 of H 37 RvMTB in concentration of ∼ 0.468 ppm. To compare with 40 ppm of rifampicin, ∼ 0.663 ppm of 5 Ag :5 ZnO had the ability to kill of H 37 RvMTB, too. Based on previous research, ZnO NPs had strong anti-tubercular impact against H 37 RvMTB to in-vitro condition, but it was toxic in concentration of ∼ 0.468 ppm to both of THP-1 and normal lung (MRC-5) cell lines. It also seems that 5 Ag :5 ZnO is justified because in concentration of ∼ 0.663 ppm of 5 Ag :5 ZnO , phagocytized H 37 RvMTB into the THP-1 had died without any toxicity effects against THP-1 and also MRC-5 cell lines. It is obvious that the mixture of colloidal silver and zinc oxide NPs with ratio of 5 Ag :5 ZnO would be trustworthy options as anti-tubercular nano-drugs in future researches. Copyright © 2017. Published by Elsevier Ltd.

Radiation protective effect of hypoxia-inducible factor-1α (HIF-1α) on human oral squamous cell carcinoma cell lines

International Nuclear Information System (INIS)

Hosokawa, Y.; Okumura, K.; Terashima, S.; Sakakura, Y.

2012-01-01

We examined the effects of 5-Gy radiation on the expression of hypoxia-inducible factor-1α (HIF-1α) and the radiosensitivity of five human oral squamous cell carcinoma (OSCC) cell lines (SAS, Ca9-22, TT, BSC-OF and IS-FOM). In all of the cell lines, HIF-1α was expressed in mRNA, and radiation had no influence on gene transcription. The number of apoptotic cells increased 72 h after irradiation in cell lines SAS, Ca9-22 and TT cells, indicating low transcriptional levels of HIF-1α, and the levels of non-cleaved caspase-3, an executioner of apoptosis, and non-cleaved poly (adenosine diphosphate-ribose) polymerase (PARP), a marker of DNA damage early in apoptosis, decreased simultaneously. Conversely, radiation failed to induce apoptosis or to decrease expression of non-cleaved caspase-3 and PARP in cell-lines BSC-OF and IS-FOM cells that expressed high levels of HIF-1α. BSC-OF and IS-FOM cells exhibited high migratory capacity. When CoCl 2 was present in the medium, HIF-1α expression increased along with the survival of Ca9-22 cells after radiation exposure. These results suggest that OSCC cells expressing high levels of HIF-1α are resistant to radiation. HIF-1α can be used to control the short term radiosensitivity of cells. (authors)

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

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Carsten Geiss

2017-10-01

Full Text Available Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

Heterogeneity in radiosensitivity within esophageal cell line CaEs-17 and amplification of H-ras gene

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Wang Shunbao; Guo Lei; Niu Wenzhe

1997-01-01

Ten clones were picked from cultured colonies of CaEs-17 cell line and developed to a subcell line. The values of SF 2 were measured for each subcell line. Two radioresistant subcell lines, clone 7 and clone 10 were established. According to survival curve assay, for wild type, the value of D 0 was 1.57 Gy, D Q was 1.07 Gy, N was 1.96, SF 2 was 0.41. For clone 7, the value of D 0 was 1.64 Gy, D Q was 1.85 Gy, N was 3.07, SF 2 was 0.53. For clone 10, the value of D 0 was 1.58 Gy, D Q was 2.04 Gy, N was 3.63, SF 2 was 0.61. Clone 7 and clone 10 have much higher values of D Q and N than those of wild type. There was amplification of H-ras gene in clone 10 after 2 Gy irradiation. The amplification of H-ras gene in clone 10 after 2 Gy irradiation might be involved in hetero geneity of CaEs-17 cell line

The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

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Wang, Ji-meng [School of Medicine, Nankai University, Tianjin 300071 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Zhao, Hong-xi [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038 (China); Wang, Li [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Gao, Zhi-ying, E-mail: gaozy301@yahoo.com.cn [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Yao, Yuan-qing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China)

2013-05-10

Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

UPregulated single-stranded DNA-binding protein 1 induces cell chemoresistance to cisplatin in lung cancer cell lines.

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Zhao, Xiang; He, Rong; Liu, Yu; Wu, Yongkai; Kang, Leitao

2017-07-01

Cisplatin and its analogues are widely used as anti-tumor drugs in lung cancer but many cisplatin-resistant lung cancer cases have been identified in recent years. Single-stranded DNA-binding protein 1 (SSDBP1) can effectively induce H69 cell resistance to cisplatin in our previous identification; thus, it is necessary to explore the mechanism underlying the effects of SSDBP1-induced resistance to cisplatin. First, SSDBP1-overexpressed or silent cell line was constructed and used to analyze the effects of SSDBP1 on chemoresistance of lung cancer cells to cisplatin. SSDBP1 expression was assayed by real-time PCR and Western blot. Next, the effects of SSDBP1 on cisplatin sensitivity, proliferation, and apoptosis of lung cancer cell lines were assayed by MTT and flow cytometry, respectively; ABC transporters, apoptosis-related genes, and cell cycle-related genes by real-time PCR, and DNA wound repair by comet assay. Low expression of SSDBP1 was observed in H69 cells, while increased expression in cisplatin-resistant H69 cells. Upregulated expression of SSDBP1 in H69AR cells was identified to promote proliferation and cisplatin resistance and inhibit apoptosis, while downregulation of SSDBP1 to inhibit cisplatin resistance and proliferation and promoted apoptosis. Moreover, SSDBP1 promoted the expression of P2gp, MRP1, Cyclin D1, and CDK4 and inhibited the expression of caspase 3 and caspase 9. Furthermore, SSDBP1 promoted the DNA wound repair. These results indicated that SSDBP1 may induce cell chemoresistance of cisplatin through promoting DNA repair, resistance-related gene expression, cell proliferation, and inhibiting apoptosis.

Thermotolerance and thermosensitization in CHO and R1H cells: a comparative study

International Nuclear Information System (INIS)

Dikomey, E.; Eickhoff, J.; Jung, H.

1984-01-01

In CHO and R1H cells thermotolerance was induced by a pre-incubation at 40 0 C, by an acute heat shock at 43 0 C followed by a time interval at 37 0 C, and during continuous heating at 42 0 C. Thermotolerance, which was tested at 43 0 , primarily causes an increase in D 0 of the heat-response curve. The degree of maximum thermotolerance was found to be generally more pronounced in CHO than in R1H cells, but the time interval at 37 0 C, as well as at 40 0 C, to reach this maximum level was the same in both cell lines. CHO and R1H cells could be sensitized to 40 0 C by a pre-treatment at 43 0 C. When compared for the same survival rate after pre-treatment at 43 0 C alone the degree of thermosensitization was about the same in both cell lines. In either cell line thermosensitization was found to be suppressed when cells were made thermotolerant by a previous incubation at 40 0 C for 16 hours. (author)

Dose-rate effects between 0.3 and 30 Gy/h in a normal and a malignant human cell line

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Amdur, R.J.; Bedford, J.S.

1994-01-01

This study used continuous open-quotes intermediateclose quotes dose rate irradiation (0.3-30 Gy/h) to compare the capacity for and repair of sublethal radiation damage in different cell lines growing in tissue culture. Two human cell lines were studied; one was derived from normal human fibroblasts (AG1522) and the other from a squamous cell carcinoma of the uterine cervix (HTB-35). Dose-response curves for clonogenic survival were determined following irradiation of plateau-phase cultures at five different dose rates: 22.6, 6.12, 3.65, 1.04, and 0.38 Gy/h. Subculture following irradiation was delayed for 8-24 h to allow for the full repair of open-quotes potentially lethal damage.close quotes A significant dose-rate effect was seen in both cell lines. For irradiation at the highest dose rate, survival at 2 Gy (SF2) and the α/β ratio were similar for the two cell lines (approximately 0.7 and 8.0 Gy, respectively) but the half-time of repair of sublethal damage was estimated to be approximately five times longer in the normal human fibroblast line (154 min) than in the carcinoma (31 min) cell line. These results indicate that measuring the dose-rate effect between 0.3 and 30 Gy/h is a useful way to identify and quantify differences in sublethal damage repair between cell lines. To the extent that in vitro and in vivo repair parameters are similar, and that representative tumor biopsy specimens can be examined in this way, this approach may provide a prospective way of determining the dose rate (brachytherapy) or fractionation schedule that will optimize the therapeutic ratio. 32 refs., 1 fig

The Expression and Biological Significance of PD-L1 on Lung Cancer Cell Lines

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Cheng CHEN

2009-08-01

Full Text Available Background and objective Tumor-associated PD-L1 expression was recently shown to promote T-cell apoptosis and proposed as a potential mechanism of immune evasion by tumors. On the basis of the ability of tumor-associated PD-L1 to mediate activated T-cell death, it is likely that manipulation of the PD-L1 pathway at defined time points during the development of the T-cell antitumor immune response can enhance the efficacy of T-cell-based immunotherapy. Here, the levels of expression of PD-L1 on lung cancer cell lines and its role in interaction of CTL and target cells was investigated. Methods Human PBMC derived DCs were loaded with apoptotic tumor cells and stimulated by CD40 mAb (5C11. Tumor specific CTL was generated in vitro by autologous T cells co-cultured with mature DCs. Expression of PD-L1 on lung cancer cell lines H1299 and A549 were analyzed by FCM. JAM assay was used to detect the cytolytic activity of CTL with or without blocking PD-L1 by PD-L1 mAb respectively. The concentrations of IFN-γ in supernatants from distinct groups were analyzed by ELISA. Results Tumor cells-loaded mature DCs could induce the generation of the tumor specific CTL. Expression of PD-L1 was low on A549 cell, but high on H1299 cell. Blockade of PD-L1 on A549 could not improve cytolytic effect of CTL on target cells and IFN-γ production, but fragmentation of H1299 cells and IFN-γ production were significantly enhanced by the combination of PD-L1 mAb and CTL. Conclusion Expression of PD-L1 on lung cancer cell line can decrease the cytolytic effect of CTL on target cells.

G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

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D'Anna, J.A.; Thayer, M.M.; Tobey, R.A.; Gurley, L.R.

1985-01-01

The authors have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [ 3 H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O 4 ; the total HC1O 4 -soluble, Cl 3 CCO 2 H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure

Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

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Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

2007-07-01

To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

Histones H10a and H10b are the same as CHO histones H1(III) and H1(IV):new features of H10 phosphorylation during the cell cycle

International Nuclear Information System (INIS)

D'Anna, J.A.; Gurley, L.R.; Becker, R.R.

1981-01-01

Two histone H1 fractions [H1(I) and H1(II) and two histone H1 0 fractions (H1 0 a and H1 0 b) have been isolated from butyrate-treated Chinese hamster (line CHO) cells by guanidine hydrochloride gradient chromatography on Bio-Rex 70 ion-exchange resin. The fractions have been identified by electrophoresis and amino acid analyses. Electrophoretic analysis of cyanogen bromide treated H1 0 in long acid-urea-polyacrylamide gels suggests that H1 0 a and H1 0 b differ, at least, within the 20-30 residue fragment(s) removed by the cyanogen bromide clevage. Shallow-gradient Bio-Rex 70 chromatography indicates that histones H1 0 a and H1 0 b are the same as the respective CHO histones, H1(III) and H1(IV). This identification and the phosphate incorporation data of Gurley et al. (1975) reveal new features about H1 0 phosphorylation: (1) following release from G 1 arrest, H1 0 a and H1 0 b become phosphorylated in late G 1 prior to DNA synthesis; (2) H1 0 a and H1 0 b are phosphorylated at similar rates throughout the cell cycle. These and other data demonstrate that histone H1 0 is phosphorylated in a cell cycle dependent fashion which mimics that of histone H1

[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

Science.gov (United States)

Wang, Lili; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Zhao, Song; Ma, Dongchu

2016-10-01

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect

Analysis of 125I-[Tyr3] octreotide receptors of NCI-H466 cell line

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Sun Junjie; Fan Wo; Xu Yujie; Zhang Youjiu; Zhu Ran

2002-01-01

Objective: To study the affinity of small cell lung carcinoma to [Tyr 3 ] octreotide (TOC). Methods: Taking 125 I-[Tyr 3 ] octreotide (labeled by chloramine-T method), as the ligand, small cell lung carcinoma NCI-H466 cell line was inspected for the receptor-binding points and affinity constant. Results: The radio-chemical purity of 125 I-TOC purified through sephadex G-10 was higher than 95%. Receptor analysis study showed that the expression of somatostatin receptors on NCI-H446 cells was numerous (Bmax = 1.17 x 10 5 /cell) with strong affinity to 125 I-TOC (Kd = 0.56 nM). Conclusion: Labeled TOC could be used for small cell lung carcinoma receptor imaging and radio-pharmaceutical therapy

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange.

Science.gov (United States)

Montrose, M H; Murer, H

1986-01-01

Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM). In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride. These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na+o/H+i exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

Investigation of hTERT gene expression levels in two cell lines infected by high-risk human papilloma virus

Directory of Open Access Journals (Sweden)

Maryam Akhtari

2016-07-01

Full Text Available Background: Human papilloma virus (HPV is one of the most important factors in cervical cancer. Viral sequences are integrated into the host cell genome. In mild cases the virus causes skin damages, in severe cases it leads to cancer. Like many other cancers, telomerase gene expression was increased in cervical cancer. This enzyme is a reverse transcriptase that contains two common subunits: i catalytic protein called human telomerase reverse transcriptase (hTERT and, ii RNA sequence called hTR. hTERT expression is hardly found in any somatic tissues. Detection of high telomerase activity in human cells, lead to tumor genesis. So hTERT can be used as a diagnostic tool in cancer detection. Methods: This experimental study was carried out from May 2013 to April 2014 in Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences in Tehran, Iran. Caski and Hela cancer cell lines were used which contain HPV16 and HPV18 respectively. Cell lines were cultured and total RNA was extracted. Following normalization agent glyceraldehyde-3-phosphate dehydrogenase (GADPH, hTERT expression level was determining by real-time PCR method. For each sample, the expression level of hTERT and GAPDH were quantified as copy numbers (per reaction using the standard curve. Finally, hTERT levels in Hela and Caski cell lines were compared quantitatively by t-test using GraphPad statistic software version 5 (San Diego, CA, USA. Results: According to the charts real-time PCR, hTERT gene expression in Hela and Caski cancer cell lines is significantly different (t=0.0319. Conclusion: All results confirm that hTERT expression levels in Hela and Caski cell lines are significantly different and the level of hTERT expression in the Caski cell line was slightly higher than that of Hela cell line. The significant difference between hTERT mRNA expression levels reported here could be used as a tumor marker for HPV16 and HPV18 in cervical cancer.

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

Science.gov (United States)

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

Science.gov (United States)

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Establishment of optimized MDCK cell lines for reliable efflux transport studies.

Science.gov (United States)

Gartzke, Dominik; Fricker, Gert

2014-04-01

Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line

DEFF Research Database (Denmark)

T. Trubitt, Rebecca; Rabeneck, D. Brett; Bujak, Joanna

2015-01-01

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1–2 days (20–80 Ω⋅cm2), which was then mai......In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1–2 days (20–80 Ω⋅cm2), which...... was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6 h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to b15% of pre-treatment level. Cortisol...... detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive...

INFRARED ABSORPTION LINES TOWARD NGC 7538 IRS 1: ABUNDANCES OF H{sub 2}, H{sub 3}{sup +}, AND CO

Energy Technology Data Exchange (ETDEWEB)

Goto, Miwa [Universitäts-Sternwarte München, Scheinerstr. 1, D-81679 Munich (Germany); Geballe, T. R. [Gemini Observatory, 670 North A‘ohoku Place, Hilo, HI 96720 (United States); Usuda, Tomonori, E-mail: mgoto@usm.lmu.de, E-mail: tgeballe@gemini.edu, E-mail: usuda@naoj.org [Subaru Telescope, 650 North A‘ohoku Place, Hilo, HI 96720 (United States)

2015-06-10

We report high-resolution near-infrared absorption spectroscopy of H{sub 2}, H{sub 3}{sup +}, and CO toward the young high mass object NGC 7538 IRS 1. The v = 1–0 H{sub 2} S(0) line and lines in the CO v = 2–0 band were detected; the v = 1–0 H{sub 2} S(1) line and the v = 1–0 H{sub 3}{sup +} lines [R(1, 1){sup l}, R(1, 0), R(1, 1){sup u}] were not detected. The line of sight traverses two clouds, with temperatures 45 and 259 K and with roughly equal column densities of CO. Assuming that H{sub 2} is at the same temperature as CO and that the two species are uniformly mixed, [H{sub 2}]/[CO] = 3600 ± 1200. NGC 7538 is the most distant object from the Galactic center for which [H{sub 2}]/[CO] has been directly measured using infrared absorption spectroscopy.

Syntheses and modulations in the chromatin contents of histones H1/sup o/ and H1 during G1 and S phases in Chinese hamsters cells

International Nuclear Information System (INIS)

D'Anna, J.A.; Gurley, L.R.; Tobey, R.A.

1982-01-01

Flow cytometry, conventional autoradiography, and autoradiography employing high concentrations of high specific activity [ 3 H]thymidine indicate that (1) treatment of Chinese hamster ovary (line CHO) cells with butyrate truly blocks cells in G 1 and (2) cells blocked in G 1 by isoleucine deprivation remain blocked in G 1 when they are released into complete medium containing butyrate. Measurements of H1/sup o/ content relative to core histones and H1/sup o/:H1 ratios indicate that H1/sup o/ is enhanced somewhat in G 1 cells arrested by isoleucine deprivation; however, (1) treatment with butyrate greatly increases the H1/sup o/ content in G 1 -blocked cells, and (2) the enhancement is very sensitive to butyrate concentration. Measurements of relative histone contents in the isolated chromatin of synchronized cultures also suggest that the acid-soluble content of histone H1 (relative to core histones) becomes greatly depleted in the isolated chromatin when synchronized cells are blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade. We also have measured [ 3 H]lysine incorporation, various protein ratios, and relative rates of deposition of newly synthesized H1/sup o/, H1, and H4 onto chromatin during G 1 and S in the absence of butyrate. The results suggest a dynamic picture of chromatin organization in which (1) newly synthesized histone H1/sup o/ binds to chromatin during traverse of G 1 and S phases and (2) histone H1 dissociates from (or becomes loosely bound to) chromatin during prolonged early S-phase block with hydroxyurea

THP-1 cell line: an in vitro cell model for immune modulation approach.

Science.gov (United States)

Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line

KAUST Repository

Benavente, Ernest Diez

2017-12-16

Plasmodium knowlesi, a common parasite of macaques, is recognized as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.

A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line

KAUST Repository

Benavente, Ernest Diez; de Sessions, Paola Florez; Moon, Robert W.; Grainger, Munira; Holder, Anthony A; Blackman, Michael J.; Roper, Cally; Drakeley, Christopher J.; Pain, Arnab; Sutherland, Colin J.; Hibberd, Martin L.; Campino, Susana; Clark, Taane G

2017-01-01

Plasmodium knowlesi, a common parasite of macaques, is recognized as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.

Analysis of CTCL cell lines reveals important differences between mycosis fungoides/Sézary syndrome vs. HTLV-1+ leukemic cell lines

DEFF Research Database (Denmark)

Netchiporouk, Elena; Gantchev, Jennifer; Tsang, Matthew

2017-01-01

HTLV-1 is estimated to affect ~20 million people worldwide and in ~5% of carriers it produces Adult T-Cell Leukemia/Lymphoma (ATLL), which can often masquerade and present with classic erythematous pruritic patches and plaques that are typically seen in Mycosis Fungoides (MF) and Sézary Syndrome...... (SS), the most recognized variants of Cutaneous T-Cell Lymphomas (CTCL). For many years the role of HTLV- 1 in the pathogenesis of MF/SS has been hotly debated. In this study we analyzed CTCL vs. HTLV-1+ leukemic cells. We performed G-banding/spectral karyotyping, extensive gene expression analysis......, TP53 sequencing in the 11 patient-derived HTLV- 1+ (MJ and Hut102) vs. HTLV-1- (Myla, Mac2a, PB2B, HH, H9, Hut78, SZ4, Sez4 and SeAx) CTCL cell lines. We further tested drug sensitivities to commonly used CTCL therapies and studied the ability of these cells to produce subcutaneous xenograft tumors...

Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

International Nuclear Information System (INIS)

D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

1980-01-01

A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

International Nuclear Information System (INIS)

Schilling, Daniela; Bayer, Christine; Geurts-Moespot, Anneke; Sweep, Fred CGJ; Pruschy, Martin; Mengele, Karin; Sprague, Lisa D; Molls, Michael

2007-01-01

Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. HIF-1α immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2 – 4 h) of hypoxic exposure (< 0.66 % O 2 ), reoxygenation (24 h, 20 % O 2 ), and radiation (0, 2, 5 and 10 Gy). HIF-1α expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. Our data suggest that both, short-term (~4 – 8 h) and long-term (~20 – 24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of

Effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69

International Nuclear Information System (INIS)

He Wenqian; Liu Zhonghua

2007-01-01

Objective: To study the effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69. Methods: Cultured NCI-H69 cells were derided into 4 groups: bcl-2 antisense oligodexynucleotides (ASODN) added, sense oligodexynucleotides (SODN) added, nonsense oligodexynucleotides (NSODN) added and control (no nucleotides added), the oligodexynucleotides were transfected into the cultured cells with oligofectamine. The cellular expression of Bcl-2 protein 72h later was examined with Western-Blot. The four different groups of cultured tumor cells were treated with etopside(Vp-16) at different concentrations (0, 0.25, 0.5, 1.0, 2.0 and 4.0 μg/ml) for 48hr then the cell survival fraction was assessed with MTY test. Results: The apoptotic rate of cells in the ASODN group was significantly higher than that of the control group, also, the survival fraction of cells in ASODN group was significantly lower than that of the control group. The Bcl-2 protein expression in ASODN group was significantly lower than that in the control group, but no inhibition was observed in SODN and NSODN groups. Conclusion: The bcl-2 ASODN could enhance the sensitivity to chemotherapy with Vp-16 in small cell lung cancer cell line NCI-H69 by effectively blocking bcl-2 gene expression. (authors)

Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines

Directory of Open Access Journals (Sweden)

Travert Carine

2010-10-01

Full Text Available Abstract Background Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. Methods The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. Results All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%. CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. Conclusion Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

International Nuclear Information System (INIS)

Puxeddu, Efisio; Zhao Guisheng; Stringer, James R.; Medvedovic, Mario; Moretti, Sonia; Fagin, James A.

2005-01-01

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10 6 cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P < 0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a plausible

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

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Puxeddu, Efisio [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Zhao Guisheng [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Stringer, James R. [Department of Molecular Genetics, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Medvedovic, Mario [Center for Biostatistic Service, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Moretti, Sonia [Dipartimento di Medicina Interna, Universita degli Studi di Perugia, Via E. dal Pozzo, Perugia 06126, (Italy); Fagin, James A. [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States)]. E-mail: james.fagin@uc.edu

2005-02-15

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10{sup 6} cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P < 0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a

Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

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Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

2011-01-01

Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

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Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level

High hRFI expression correlates with resistance to Fluoro pyrimidines in human colon cancer cell lines and in xenografts

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Sasaki, S.; Tokyo Univ., Tokyo; Watanabe, T.; Konishi, T.; Kitayama, J.; Nagawa, H.; Kobunai, T.

2005-01-01

We previously reported that the over-expression of hRFI, a protein preferentially expressed in the digestive tract regions of several cancers, exhibited a tendency to inhibit TNF-α induced apoptosis. In this study, we sought to determine the potential effect of hRFI expression on the sensitivity to 5-fluorouracil (5-FU) and/or other fluoro pyrimidines. For the whole lysates of 8 colon cancer cell lines, we performed Western blotting with anti-hRFI antibody and analyzed the correlations between the expression level of hRFI and the cell lines' sensitivity to 5-FU induced apoptosis. Furthermore, for a tissue micro array consisting of 32 xenograft derived human cancer cell lines, we examined the expression levels of hRFI and survivin by immunohistochemical staining, and analyzed the correlations between the expression of each protein and the sensitivity to several chemotherapeutic agents in the xenografts examined. Both in colon cancer cell lines and in xenografts, the expression level of hRFI was correlated with resistance to 5-FU and its derivatives. This evidence suggests that hRFI may be a marker predicting the response to fluorouracil derived chemotherapeutic agents and that the reduction of the expression level of hRFI might improve the outcome of chemotherapy

Morphometric studies with attached mouse C3H/10T 1/2 cells

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Geard, C.R.; Harding, T.

1981-01-01

Studies of in vitro transformation using the Syrian hamster embryo cell system and the mouse C3H/10T 1/2 cell system form an integral part of this laboratory's activities. As part of the studies with the mouse cell line we have monitored the behavior of these cells in culture in order to ascertain those variables which might influence the expression of transformation. The study of transformed cells versus normal cells could lead to insight into an earlier definition of transformation that the clonal morphological change currently in use. This present report details the changes in cellular morphology with time in culture of normal mouse C3H/10T 1/2 cells from early passages (9 to 13) and x-ray transformed cells which have been maintained in culture for three years

Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

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Norris, J S; Kohler, P O

1978-01-01

Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

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Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

Generation of an ASS1 heterozygous knockout human embryonic stem cell line, WAe001-A-13, using CRISPR/Cas9

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Fang Yuan

2018-01-01

Full Text Available The ASS1 gene encodes argininosuccinate synthetase-1, a cytosolic enzyme with a critical role in the urea cycle. Mutations are found in all ASS1 exons and cause the autosomal recessive disorder citrullinemia. Using CRISPR/Cas9-editing, we established the WAe001-A-13 cell line, which was heterozygous for an ASS1 mutation, from the human embryonic stem cell line H1. The WAe001-A-13 cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

Induction of Programmed Cell Death by Parvovirus H-1 in U937 Cells: Connection with the Tumor Necrosis Factor Alpha Signalling Pathway

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Rayet, Béatrice; Lopez-Guerrero, José-Antonio; Rommelaere, Jean; Dinsart, Christiane

1998-01-01

The human promonocytic cell line U937 undergoes apoptosis upon treatment with tumor necrosis factor alpha (TNF-α). This cell line has previously been shown to be very sensitive to the lytic effect of the autonomous parvovirus H-1. Parvovirus infection leads to the activation of the CPP32 ICE-like cysteine protease which cleaves the enzyme poly(ADP-ribose)polymerase and induces morphologic changes that are characteristic of apoptosis in a way that is similar to TNF-α treatment. This effect is also observed when the U937 cells are infected with a recombinant H-1 virus which expresses the nonstructural (NS) proteins but in which the capsid genes are replaced by a reporter gene, indicating that the induction of apoptosis can be assigned to the cytotoxic nonstructural proteins in this cell system. The c-Myc protein, which is overexpressed in U937 cells, is rapidly downregulated during infection, in keeping with a possible role of this product in mediating the apoptotic cell death induced by H-1 virus infection. Interestingly, four clones (designated RU) derived from the U937 cell line and selected for their resistance to H-1 virus (J. A. Lopez-Guerrero et al., Blood 89:1642–1653, 1997) failed to decrease c-Myc expression upon treatment with differentiation agents and also resisted the induction of cell death after TNF-α treatment. Our data suggest that the RU clones have developed defense strategies against apoptosis, either by their failure to downregulate c-Myc and/or by activating antiapoptotic factors. PMID:9765434

Ionizing radiation effects on the KG1A primitive hematopoietic cell line

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Clave, Emmanuel; Carosella, Edgardo D.; Gluckman, Eliane; Dubray, Bernard; Socie, Gerard

1996-01-01

Purpose: Better understanding of radiation-induced effects on the hematopoietic system is important in both the context of therapeutic intervention and accidental exposure. However, direct study of these effects on the hematopoietic stem cell pool is hampered by the small number of accessible cells. We, thus, studied radiation-induced effects on the KG1a stem cell line. Methods and Materials: We confirmed and extended the immunophenotype of KG1a with monoclonal antibodies, established a radiation survival curve, and quantified mRNAs by Northern blotting 30 min after 1, 2, and 3 Gy of ionizing radiation (IR) and followed for up to 48 h after a 3 Gy dose. Cell cycle status and apoptosis were assessed by fluorescent-activated cell sorter (FACS) analysis, cell morphology, and DNA fragmentation. Results: KG1a was found to be CD34+, CD7+, Thy1 low, CD38 low, lineage negative (neg), C-KITneg and HLA-DRneg, a phenotype consistent with a primitive hematopoietic origin. This immunophenotype was not altered by x-ray irradiation. The D 0 value was 1.75 Gy. We showed a time-dependent variation of c-jun mRNA expression with an early and transient dose-dependent induction followed by a second increase at 24 and 48 h: a biphasic dose-dependent variation of bcl-2 expression 30 min after irradiation with a reduction of mRNA level at 1 Gy, and a normalization at higher doses and stable levels of mRNA for c-fos, c-myc, G-CSF, GM-CSF, IL-6, TNF-α, TGF-β, and MIP-1α genes. Cell cycle analysis showed the absence of G1/S phase arrest, a point consistent with the absence of detection of P53 mRNA by Northern blot analysis. The dose-dependent G2/M phase arrest was not followed by significant apoptotic cell death. Conclusion: Taken together, this data indicates that radiation-induced cell death of KG1a, a cell line that has a relatively high D 0 value, does not seem to be the result of the apoptotic pathway but occurs subsequent to a G2/M phase arrest

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

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van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines

DEFF Research Database (Denmark)

Christensen, C R; Klingelhöfer, Jörg; Tarabykina, S

1998-01-01

identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse...

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

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Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation

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Aušra Liubavičiūtė

2015-11-01

Conclusions: Our data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24 h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72 h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72 h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

International Nuclear Information System (INIS)

Tátrai, Péter; Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Buchan, Gyöngyi; Mádi, András; Uher, Ferenc

2012-01-01

Highlights: ► We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. ► hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. ► SV40T introduced along with hTERT abrogates proliferation control and multipotency. ► hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC hTERT , ASC Bmi-1 , ASC Bmi-1+hTERT and ASC SV40T+hTERT were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC Bmi-1 had limited replicative potential, while the rapidly proliferating ASC SV40T+hTERT acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC hTERT and ASC hTERT+Bmi-1 , on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC hTERT also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC hTERT are prone to transformation during extensive

ABCC5, ERCC2, XPA and XRCC1 transcript abundance levels correlate with cisplatin chemoresistance in non-small cell lung cancer cell lines

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Khuder Sadik A

2005-05-01

Full Text Available Abstract Background Although 40–50% of non-small cell lung cancer (NSCLC tumors respond to cisplatin chemotherapy, there currently is no way to prospectively identify potential responders. The purpose of this study was to determine whether transcript abundance (TA levels of twelve selected DNA repair or multi-drug resistance genes (LIG1, ERCC2, ERCC3, DDIT3, ABCC1, ABCC4, ABCC5, ABCC10, GTF2H2, XPA, XPC and XRCC1 were associated with cisplatin chemoresistance and could therefore contribute to the development of a predictive marker. Standardized RT (StaRT-PCR, was employed to assess these genes in a set of NSCLC cell lines with a previously published range of sensitivity to cisplatin. Data were obtained in the form of target gene molecules relative to 106 β-actin (ACTB molecules. To cancel the effect of ACTB variation among the different cell lines individual gene expression values were incorporated into ratios of one gene to another. Each two-gene ratio was compared as a single variable to chemoresistance for each of eight NSCLC cell lines using multiple regression. In an effort to validate these results, six additional lines then were evaluated. Results Following validation, single variable models best correlated with chemoresistance (p ERCC2/XPC, ABCC5/GTF2H2, ERCC2/GTF2H2, XPA/XPC and XRCC1/XPC. All single variable models were examined hierarchically to achieve two variable models. The two variable model with the highest correlation was (ABCC5/GTF2H2, ERCC2/GTF2H2 with an R2 value of 0.96 (p Conclusion These results provide markers suitable for assessment of small fine needle aspirate biopsies in an effort to prospectively identify cisplatin resistant tumors.

Anti-tumor effect of bisphosphonate (YM529 on non-small cell lung cancer cell lines

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Date Hiroshi

2007-01-01

Full Text Available Abstract Background YM529 is a newly developed nitrogen-containing bisphosphonate (BP classified as a third-generation BP that shows a 100-fold greater potency against bone resorption than pamidronate, a second-generation BP. This agent is, therefore expected to be extremely useful clinically for the treatment of osteoporosis and hypercalcemia. Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both in vitro or/and in vivo. In this study, we investigate the anti-tumor effect of YM529 on non-small cell lung cancer (NSCLC. Methods Direct anti-tumor effect of YM529 against 8 NSCLC cell lines (adenocarcinoma: H23, H1299, NCI-H1819, NCI-H2009, H44, A549, adenosquamous cell carcinoma: NCI-H125, squamous cell carcinoma: NCI-H157 were measured by MTS assay and calculated inhibition concentration 50 % (IC50 values. YM529 induced apoptosis of NCI-H1819 was examined by DNA fragmentation of 2 % agarose gel electrophoresis and flowcytometric analysis (sub-G1 method. We examined where YM529 given effect to apoptosis of NSCLC cells in signaling pathway of the mevalonate pathway by western blotting analysis. Results We found that there was direct anti-tumor effect of YM529 on 8 NSCLC cell lines in a dose-dependent manner and their IC50 values were 2.1 to 7.9 μM and YM529 induced apoptosis and G1 arrest cell cycle with dose-dependent manner and YM529 caused down regulation of phospholyration of ERK1/2 in signaling pathways of NSCLC cell line (NCI-H1819. Conclusion Our study demonstrate that YM529 showed direct anti-tumor effect on NSCLC cell lines in vitro, which supports the possibility that third-generation BPs including YM529 can be one of therapeutic options for NSCLC.

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Investigation of internalization and cytotoxicity of 125I-[Tyr3]-octreotide in NCI-H446 cell line

International Nuclear Information System (INIS)

Sun Junjie; Fan Wo; Xu Yujie; Zhang Youjiu; Zhu Ran; Hu Mingjiang

2004-01-01

Objective: To investigate the [Tyr 3 ]-octreotide (TOC) internalizing capacity of NCI-H446 cell line, and the cytotoxicity of 125 I-TOC in NCI-H446 cell line. To assess the therapeutic radiopharmaceutical potentiality of 125 I-TOC for the somatostatin receptor (SSTR) positive tumor. Methods: NCI-H446 cells were incubated together with 125 I-TOC for different periods of time, the amount of internalized 125 I-TOC and the 125 I-TOC bound on the cellular nucleus were detected with γ counter, respectively. The viability of the cells was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points with various doses of 125 I-TOC, free 125 I and TOC. Results: 125 I-TOC was internalized into the nucleus and bound on the nucleus in a time-dependent manner. 125 I-TOC bound on the nucleus increased to the highest level at 24 h, the amount of nucleus bound 125 I-TOC at 24 h was 7 times higher than that at 0.5 h. Cytotoxicity of 125 I-TOC in SSTR positive NCI-H446 cells was also dose- and time-dependent. The supreme effect of cytotoxicity was found at 96 h with 74 kBq 125 I-TOC, the survival ratio of cells was reduced to (44.8 ± 7.2)%. Conclusions: 125 I-TOC can be internalized into SSTR positive cells mediated by SSTR. The NCI-H446 cells can be killed by Auger electron emitting from 125 I-TOC. Effect of cytotoxicity showed dose- and time-dependent

Characterisation of the cell line HC-AFW1 derived from a pediatric hepatocellular carcinoma.

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Sorin Armeanu-Ebinger

Full Text Available Current treatment of paediatric hepatocellular carcinoma (HCC is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP, Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

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Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

2017-09-01

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Efficient Generation of NKX6-1+ Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines

Directory of Open Access Journals (Sweden)

M. Cristina Nostro

2015-04-01

Full Text Available Human pluripotent stem cells (hPSCs represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10, and inhibitors of bone morphogenetic protein (BMP and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

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Dastjerdi, M Nikbakht; Babazadeh, Z; Rabbani, M; Gharagozloo, M; Esmaeili, A; Narimani, M

2014-01-01

Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 μM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (pPANC-1 through p21 and Bax pathway but not through RASSF1A.

Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

International Nuclear Information System (INIS)

Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

2003-01-01

Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

Enantioselective kappa opioid binding sites on the macrophage cell line, P388d sub 1

Energy Technology Data Exchange (ETDEWEB)

Carr, D.J.J.; Blalock, J.E. (Univ. of Alabama, Birmingham (USA)); DeCosta, B.R.; Jacobson, A.E.; Rice, K.C. (NIDDK, NIH, Bethesda, MD (USA))

1991-01-01

A kappa opioid binding site has been characterized on the macrophage cell line, P388d{sub 1}, using the kappa selective affinity ligand, ({sup 3H}(1S,2S)-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-phrrolidinyl) cyclohexyl) benzeneacetamide ((-)BD166). The kappa site has a relative molecular mass (Mr) of 38,000 under nonreducing conditions and 42,000 under reducing conditions. Moreover, it exhibits enantioselectivity in that 1S,2S-(-)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((-)-U-50,488) blocks ({sup 3}H)95{alpha},7{alpha},8{beta})-(-)-N-methyl-N-(7-(1- pyrrolidinyl)-1-oxaspiro-(4,5)-dec-8-yl)benzeneacetamide (U-69,593) binding to P388d{sub 1} cells with an IC{sub 50} = 7.0 nM whereas 1R,2R-(+)-trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl) benzeneacetamide ((+)U-50,488) blocks ({sup 3}H)U-69,593 binding to P388d{sub 1} cells with an IC{sub 50} = 700 nM.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

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Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

2012-05-25

Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

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Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

2013-01-01

Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line.

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Zhao, Jun-Xia; Zhang, Qing-Shuang; Chen, Ying; Yao, Sheng-Jie; Yan, Yong-Xin; Wang, Ying; Zhang, Jin-Xiu; Wang, Li-An

2016-05-20

The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes (apoptosis and necrosis) were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L). After treatment of different concentrations of iso-suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin (16.70%, 35.54%, and 49.20%, respectively, all P iso-suillin could induce H446 cell apoptosis through the mitochondrial pathway and the death-receptor pathway. Therefore, iso-suillin might have a potential application as a novel drug for lung cancer treatment.

Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury

International Nuclear Information System (INIS)

Bhakta, Kushal Y.; Jiang, Weiwu; Couroucli, Xanthi I.; Fazili, Inayat S.; Muthiah, Kathirvel; Moorthy, Bhagavatula

2008-01-01

Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0-72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2-4 fold), and mRNA levels (30-40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4-72 h), showed marked (2-6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD

Histone h1 depletion impairs embryonic stem cell differentiation.

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Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

In vitro synergistic antitumor efficacy of sequentially combined chemotherapy/icotinib in non‑small cell lung cancer cell lines.

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Wang, Min-Cong; Liang, Xuan; Liu, Zhi-Yan; Cui, Jie; Liu, Ying; Jing, Li; Jiang, Li-Li; Ma, Jie-Qun; Han, Li-Li; Guo, Qian-Qian; Yang, Cheng-Cheng; Wang, Jing; Wu, Tao; Nan, Ke-Jun; Yao, Yu

2015-01-01

The concurrent administration of chemotherapy and epidermal growth factor receptor‑tyrosine kinase inhibitors (EGFR‑TKIs) has previously produced a negative interaction and failed to confer a survival benefit to non‑small cell lung cancer (NSCLC) patients compared with first‑line cytotoxic chemotherapy. The present study aimed to investigate the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib in NSCLC cell lines and clarify the underlying mechanisms. HCC827, H1975, H1299 and A549 human NSCLC cell lines with wild‑type and mutant EGFR genes were used as in vitro models to define the differential effects of various schedules of cisplatin/paclitaxel with icotinib treatments on cell growth, proliferation, cell cycle distribution, apoptosis, and EGFR signaling pathway. Sequence‑dependent antiproliferative effects differed among the four NSCLC cell lines, and were not associated with EGFR mutation, constitutive expression levels of EGFR or downstream signaling molecules. The antiproliferative effect of cisplatin plus paclitaxel followed by icotinib was superior to that of cisplatin or paclitaxel followed by icotinib in the HCC827, H1975, H1299 and A549 cell lines, and induced more cell apoptosis and G0/G1 phase arrest. Cisplatin and paclitaxel significantly increased the expression of EGFR phosphorylation in the HCC827 cell line. However, only paclitaxel increased the expression of EGFR phosphorylation in the H1975 cell line. Cisplatin/paclitaxel followed by icotinib influenced the expression of p‑EGFR and p‑AKT, although the expression of p‑ERK1/2 remained unchanged. The results suggest that the optimal schedule of the combined treatment of cisplatin/paclitaxel and icotinib differed among the NSCLC cell lines. The results also provide molecular evidence to support clinical treatment strategies for NSCLC patients.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

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Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Bifenthrin activates homotypic aggregation in human T-cell lines.

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Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

2006-03-01

Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

Establishment and characterization of new cell lines of anaplastic pancreatic cancer, which is a rare malignancy: OCUP-A1 and OCUP-A2

International Nuclear Information System (INIS)

Miura, Kotaro; Kimura, Kenjiro; Amano, Ryosuke; Yamazoe, Sadaaki; Ohira, Go; Murata, Akihiro; Nishio, Kohei; Hasegawa, Tsuyoshi; Yashiro, Masakazu; Nakata, Bunzo; Ohira, Masaichi; Hirakawa, Kosei

2016-01-01

Anaplastic pancreatic cancer (APC) cell lines have been scarcely established. The morphology, gene expressions, karyotyping and epithelial-mesenchymal transition markers of newly established APC cell lines OCUP-A1 and OCUP-A2 were analyzed. Their abilities of proliferation under normoxia and hypoxia, migration and invasion were compared to 4 commercially available pancreatic ductal adenocarcinoma (PDA) cell lines. Their induction of angiogenesis, stem-like cell population and subcutaneous tumor growth in nude mice were estimated, comparing 2 PDA cell lines examined here. OCUP-A1 and OCUP-A2 cells continuously grew with spindle and polygonal shapes, respectively. Gene analysis revealed 9 gene mutations including KRAS and TP53. Karyotyping clarified numerical structural abnormalities in both cells. Loss of E-cadherin and expression of vimentin in both cell lines were observed. The doubling time of both cell lines was approximately 20 h. Proliferation, migration and invasion abilities were not notable compared to other PDA cell lines. However stem-like cell population of both cell lines was superior to a part of PDA cell lines. Moreover OCUP-A1 showed stronger hypoxia tolerance and induction of angiogenesis than other PDA cell lines. The tumorigenicity in vivo of OCUP-A2 was stronger than conventional PDA cell lines. The OCUP-A1 and OCUP-A2 cell lines of rare malignancies might be useful for investigating the biology of pancreatic cancer

Regulation of the angiopoietin-2 gene by hCG in ovarian cancer cell line OVCAR-3.

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Pietrowski, D; Wiehle, P; Sator, M; Just, A; Keck, C

2010-05-01

Angiogenesis is a crucial step in growing tissues including many tumors. It is regulated by pro- and antiangiogenic factors including the family of angiopoietins and their corresponding receptors. In previous work we have shown that in human ovarian cells the expression of angiopoietin 2 (ANG2) is regulated by human chorionic gonadotropin (hCG). To better understand the mechanisms of hCG-dependent regulation of the ANG2-gene we have now investigated upstream regulatory active elements of the ANG2-promoter in the ovarian carcinoma cell line OVCAR-3. We cloned several ANG2-promoter-fragments of different lengths into a luciferase reporter-gene-vector and analyzed the corresponding ANG2 expression before and after hCG stimulation. We identified regions of the ANG2-promoter between 1 048 bp and 613 bp upstream of the transcriptional start site where hCG-dependent pathways promote a significant downregulation of gene expression. By sequence analysis of this area we found several potential binding sites for transcription factors that are involved in regulation of ANG2-expression, vascular development and ovarian function. These encompass the forkhead family transcription factors FOXC2 and FOXO1 as well as the CCAAT/enhancer binding protein family (C/EBP). In conclusion, we have demonstrated that the regulation of ANG2-expression in ovarian cancer cells is hCG-dependent and we suggest that forkhead transcription factor and C/EBP-dependent pathways are involved in the regulation of ANG2-expression in ovarian cancer cells. Georg Thieme Verlag KG Stuttgart-New York.

Expression of G-protein inwardly rectifying potassium channels (GIRKs in lung cancer cell lines

Directory of Open Access Journals (Sweden)

Schuller Hildegard M

2005-08-01

Full Text Available Abstract Background Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1 in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. Methods GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU assay. Results GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with β2-adrenergic antagonist ICI 118,551 (100 μM daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2 was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4 mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4 mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

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Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

International Nuclear Information System (INIS)

Chromik, Ansgar M; Weyhe, Dirk; Mittelkötter, Ulrich; Uhl, Waldemar; Hahn, Stephan A; Daigeler, Adrien; Flier, Annegret; Bulut, Daniel; May, Christina; Harati, Kamran; Roschinsky, Jan; Sülberg, Dominique

2010-01-01

The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis

Detection of Apoptosis and Necrosis in Normal Human Lung Cells Using 1H NMR Spectroscopy

Science.gov (United States)

Shih, Chwen-Ming; Ko, Wun-Chang; Yang, Liang-Yo; Lin, Chien-Ju; Wu, Jui-Sheng; Lo, Tsui-Yun; Wang, Shwu-Huey; Chen, Chien-Tsu

2005-05-01

This study aimed to detect apoptosis and necrosis in MRC-5, a normal human lung cell line, by using noninvasive proton nuclear magnetic resonance (1H NMR). Live MRC-5 cells were processed first for 1H NMR spectroscopy; subsequently their types and the percentage of cell death were assessed on a flow cytometer. Cadmium (Cd) and mercury (Hg) induced apoptosis and necrosis in MRC-5 cells, respectively, as revealed by phosphatidylserine externalization on a flow cytometer. The spectral intensity ratio of methylene (CH2) resonance (at 1.3 ppm) to methyl (CH3) resonance (at 0.9 ppm) was directly proportional to the percentage of apoptosis and strongly and positively correlated with PI staining after Cd treatment (r2 = 0.9868, P In contrast, this ratio only increased slightly within 2-h Hg treatment, and longer Hg exposure failed to produce further increase. Following 2-h Hg exposure, the spectral intensity of choline resonance (at 3.2 ppm) was abolished, but this phenomenon was absent in Cd-induced apoptosis. These findings together demonstrate that 1H NMR is a novel tool with a quantitative potential to distinguish apoptosis from necrosis as early as the onset of cell death in normal human lung cells.

Temperature sensitivity on growth and/or replication of H1N1, H1N2 and H3N2 influenza A viruses isolated from pigs and birds in mammalian cells.

Science.gov (United States)

Massin, Pascale; Kuntz-Simon, Gaëlle; Barbezange, Cyril; Deblanc, Céline; Oger, Aurélie; Marquet-Blouin, Estelle; Bougeard, Stéphanie; van der Werf, Sylvie; Jestin, Véronique

2010-05-19

Influenza A viruses have been isolated from a wide range of animal species, aquatic birds being the reservoir for their genetic diversity. Avian influenza viruses can be transmitted to humans, directly or indirectly through an intermediate host like pig. This study aimed to define in vitro conditions that could prove useful to evaluate the potential of influenza viruses to adapt to a different host. Growth of H1N1, H1N2 and H3N2 influenza viruses belonging to different lineages isolated from birds or pigs prior to 2005 was tested on MDCK or NPTr cell lines in the presence or absence of exogenous trypsin. Virus multiplication was compared at 33, 37 and 40 degrees C, the infection site temperatures in human, swine and avian hosts, respectively. Temperature sensitivity of PB2-, NP- and M-RNA replication was also tested by quantitative real-time PCR. Multiplication of avian viruses was cold-sensitive, whatever cell type. By contrast, temperature sensitivity of swine viruses was found to depend on the virus and the host cell: for an H1N1 swine isolate from 1982, multiplication was cold-sensitive on NPTr cells and undetectable at 40 degrees C. From genetic analyses, it appears that temperature sensitivity could involve other residues than PB2 residue 627 and could affect other steps of the replication cycle than replication. Copyright 2009 Elsevier B.V. All rights reserved.

Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

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Yanxing Han

2008-07-01

Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts

International Nuclear Information System (INIS)

Crompton, N.E.A.; Emery, G.C.; Shi Yuquan; Sigg, M.; Blattmann, H.

1998-01-01

We investigated various phenotypic characteristics of radiation-induced morphologically transformed C3H 10T1/2 mouse fibroblasts. The cells were treated with 8 Gy x-rays, and type II/III foci were isolated. Cell lines were developed from these foci, and subsequently clones were established from these focal lines. The clones were examined for DNA content, radiosensitivity and inducible cell cycle arrests. Besides the morphological changes associated with the transformed state, the major difference between the isolated focal lines or derived clones and the parental C3H 10T1/2 line was one of ploidy. The transformed cells often displayed aneuploid and multiple polyploid populations. No change in the radiosensitivity of the transformed cells was observed. Furthermore, the two major radiation- and staurosporine-induced G1 and G2 cell cycle arrests observed in the parental cell line were also observed in the morphological transformants, suggesting that checkpoint function was normal. (orig.)

Anticancer drugs and the regulation of Hedgehog genes GLI1 and PTCH1, a comparative study in nonmelanoma skin cancer cell lines

DEFF Research Database (Denmark)

Olesen, Uffe H; Bojesen, Sophie; Gehl, Julie

2017-01-01

Nonmelanoma skin cancer is the most common cancer in humans, comprising mainly basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC proliferation is highly dependent on the Hedgehog signaling pathway. We aimed to investigate a panel of anticancer drugs with known activity against skin...... of immortalized keratinocytes (HaCaT), BCC (UWBCC1 and BCC77015), and SCC (A431 and SCC25) cell lines. The impact of treatment on the regulation of Hedgehog pathway target genes (GLI1 and PTCH1), measured by real-time PCR, was compared between UWBCC1 and HaCaT. Varying cell line sensitivity profiles...... to the examined anticancer drugs were observed. Generally, 24-h drug exposure was sufficient to reduce cell viability. We found that 5-FU, MTX, and cisplatin significantly downregulated the expression of two genes controlled by the Hedgehog pathway (≤25-, 2.9-, and 12.5-fold, respectively, for GLI1 in UWBCC1...

Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines

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Ding-Kun Kang

Full Text Available Abstract Background: Local anesthetics (LAs are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001, concentration- (p < 0.001, and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Science.gov (United States)

Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

DEFF Research Database (Denmark)

Nielsen, C U; Amstrup, J; Steffansen, B

2001-01-01

(max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P PepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control......) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane....

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR

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Zheng Wang

2016-09-01

Full Text Available Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting

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So-Jung Kim

2017-03-01

Full Text Available Kelch-like ECH-associated protein 1 (keap1 is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a. Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC. The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential.

GLP-1 Amidation Efficiency Along the Length of the Intestine in Mice, Rats and Pigs and in GLP-1 Secreting Cell Lines

DEFF Research Database (Denmark)

Kuhre, Rune Ehrenreich; Albrechtsen, Nicolai Jacob Wewer; Windeløv, Johanne Agerlin

2014-01-01

and whether this varied with the six different locations. We also analyzed the amidation in 3 GLP-1 secreting cell lines (GLUTag, NCI-H716 and STC-1). To our surprise there were marked differences between the 3 species with respect to the concentration of GLP-1 in gut. In the mouse, concentrations increased...... sites, whereas rats and pigs on average had around 2.5 and 4 times higher levels of amidated compared to non-amidated GLP-1, although the ratio varied depending upon the location. GLUTag, NCI-H716 and STC-1 cells all exhibited partial amidation with 2-4 times higher levels of amidated compared to non...

Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro

OpenAIRE

Carsten Geiss; Zoltán Kis; Barbara Leuchs; Monika Frank-Stöhr; Jörg R. Schlehofer; Jean Rommelaere; Christiane Dinsart; Jeannine Lacroix

2017-01-01

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts...

On-line data analysis and monitoring for H1 drift chambers

International Nuclear Information System (INIS)

Duellmann, D.

1992-01-01

The on-line monitoring, slow control and calibration of the H1 central jet chamber uses a VME multiprocessor system to perform the analysis and a connected Macintosh computer as graphical interface to the operator on shift. Tasks of this system are: Analysis of event data including on-line track search; on-line calibration from normal events and testpulse events; control of the high voltage and monitoring of settings and currents; monitoring of temperature, pressure and mixture of the chambergas. A program package is described which controls the dataflow between data aquisition, different VME CPUs and Macintosh. It allows to run off-line style programs for the different tasks. (orig.)

On-line data analysis and monitoring for H1 drift chambers

Science.gov (United States)

Düllmann, Dirk

1992-05-01

The on-line monitoring, slow control and calibration of the H1 central jet chamber uses a VME multiprocessor system to perform the analysis and a connected Macintosh computer as graphical interface to the operator on shift. Task of this system are: - analysis of event data including on-line track search, - on-line calibration from normal events and testpulse events, - control of the high voltage and monitoring of settings and currents, - monitoring of temperature, pressure and mixture of the chambergas. A program package is described which controls the dataflow between data aquisition, differnt VME CPUs and Macintosh. It allows to run off-line style programs for the different tasks.

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Institute of Scientific and Technical Information of China (English)

Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

2011-01-01

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

Derivation of the human embryonic stem cell line RCM1

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P.A. De Sousa

2016-03-01

Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC?1

OpenAIRE

Fujita, Mayumi; Imadome, Kaori; Imai, Takashi

2017-01-01

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC?1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC?1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC?1 cells, metabolome analysis of the invaded PANC?1 compared with the whole cultured PANC?1 was performed using CE?TOFMS, and concentrations of 110 met...

6-Nitro-2-(3-hydroxypropyl-1H-benz[de]isoquinoline-1,3-dione, a potent antitumor agent, induces cell cycle arrest and apoptosis

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Singh Shashank K

2010-12-01

Full Text Available Abstract Background Anticancer activities of several substituted naphthalimides (1H-benz[de]isoquinoline-1,3-diones are well documented. Some of them have undergone Phase I-II clinical trials. Presently a series of ten N-(hydroxyalkyl naphthalimides (compounds 1a-j were evaluated as antitumor agents. Methods Compounds 1a-j were initially screened in MOLT-4, HL-60 and U-937 human tumor cell lines and results were compared with established clinical drugs. Cytotoxicities of compounds 1d and 1i were further evaluated in a battery of human tumor cell lines and in normal human peripheral blood mononuclear cells. Cell cycle analysis of compound 1i treated MOLT-4 cells was studied by flow cytometry. Its apoptosis inducing effect was carried out in MOLT-4 and HL-60 cells by flow cytometry using annexin V-FITC/PI double staining method. The activities of caspase-3 and caspase-6 in MOLT-4 cells following incubation with compound 1i were measured at different time intervals. Morphology of the MOLT-4 cells after treatment with 1i was examined under light microscope and transmission electron microscope. 3H-Thymidine and 3H-uridine incorporation in S-180 cells in vitro following treatment with 8 μM concentration of compounds 1d and 1i were studied. Results 6-Nitro-2-(3-hydroxypropyl-1H-benz[de]isoquinoline-1,3-dione (compound 1i, has exhibited maximum activity as it induced significant cytotoxicity in 8 out of 13 cell lines employed. Interestingly it did not show any cytotoxicity against human PBMC (IC50 value 273 μM. Cell cycle analysis of compound 1i treated MOLT-4 cells demonstrated rise in sub-G1 fraction and concomitant accumulation of cells in S and G2/M phases, indicating up-regulation of apoptosis along with mitotic arrest and/or delay in exit of daughter cells from mitotic cycle respectively. Its apoptosis inducing effect was confirmed in flow cytometric study in MOLT-4 and the action was mediated by activation of both caspase 3 and 6. Light and

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations

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Shailesh Kumar Gupta

2018-05-01

Full Text Available Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies. Keywords: Human induced pluripotent stem cells, NKX6.1, Reporter cell line, Directed differentiation, hiPSC-derived beta cells

Novel 2,3-Dihydro-1H-pyrrolo[3,2,1-ij]quinazolin-1-ones: Synthesis and Biological Evaluation

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Malose J. Mphahlele

2016-12-01

Full Text Available Herein we describe the synthesis and evaluation of a series of novel 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinazolin-1-ones for in vitro cytotoxicity against three human cancer cell lines as well as for potential antimalarial activity against the chloroquine-sensitive strain 3D7 of Plasmodium falciparum. The title compounds were prepared via PdCl2-mediated endo-dig cyclization of 2-aryl-8-(arylethynyl-6-bromo-2,3-dihydroquinazolin-4(1H-ones. The latter were prepared, in turn, via initial Sonogashira cross-coupling of 2-amino-5-bromo-3-iodobenzamide with aryl acetylenes followed by boric acid-mediated cyclocondensation of the intermediate 2-amino-3-(arylethynyl-5-bromobenzamides with benzaldehyde derivatives. The 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinazolin-1-ones 4a–k were evaluated for potential in vitro cytotoxicity against the breast (MCF-7, melanoma (B16 and endothelioma (sEnd.2 cell lines. All of the compounds except 4h and 4i were found to be inactive against the three cancer cell lines. Compound 4h substituted with a 4-methoxyphenyl and 4-fluorophenyl groups at the 3- and 5-positions was found to exhibit significant cytotoxicity against the three cancer cell lines. The presence of phenyl and 3-chlorophenyl groups at the 3- and 5-posiitons of the pyrroloquinazolinone 4i, on the other hand, resulted in significant cytotoxicity against vascular tumour endothelial cells (sEnd.2, but reduced activity against the melanoma (B16 and breast cancer (MCF-7 cells except at higher concentrations. The 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinazolin-1-ones 4a–l were found to be inactive against the chloroquine sensitive 3D7 strain of Plasmodium falciparum.

Cytotoxic effect of achatinin(H) (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7).

Science.gov (United States)

Dharmu, Indra; Ramamurty, N; Kannan, Ramalingam; Babu, Mary

2007-01-01

The hemolymph-derived achatinin(H) (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC(50) values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin(H) ranged from 6 to 10 microg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 microg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin(H) treatment. The specificity and purity of the achatinin(H) was confirmed by the Western blot assay. Achatinin(H) binding to MCF7 cells was detected by anti-achatinin(H), and visualization of the achatinin(H) binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin(H) binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin(H) treatment. The cells were arrested in G(2)/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis.

Data on the construction of a recombinant HEK293 cell line overexpressing hERG potassium channel and examining the presence of hERG mRNA and protein expression

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Yi Fan Teah

2017-10-01

Full Text Available The data presented in this article are related to the research article entitled “The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr and human ether-a-go-go-related gene (hERG expression” (Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan [1], which the possible hERG blocking properties of deoxyelephantopin were investigated. This article describes the construction of human embryonic kidney 293 (HEK293 cells overexpressing HERG potassium channel and verification of the presence of hERG mRNA and protein expression in this recombinant cell line.

Radiation Response of Cancer Stem-Like Cells From Established Human Cell Lines After Sorting for Surface Markers

International Nuclear Information System (INIS)

Al-Assar, Osama; Muschel, Ruth J.; Mantoni, Tine S.; McKenna, W. Gillies; Brunner, Thomas B.

2009-01-01

Purpose: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. Methods and Materials: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (γ-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. Results: The quantity of CSLC was generally in good agreement with primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual γ-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. Conclusions: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps"

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Lemieux Bruno

2010-12-01

Full Text Available Abstract Background In cancer cells the three-dimensional (3D telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H to diagnostic multinuclear Reed-Sternberg (RS cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei". Results Here we analyze the 3D telomere dynamics by Q-FISH in the novel Hodgkin cell line U-HO1 and its non-receptor protein-tyrosine phosphatase N1 (PTPN1 stable transfectant U-HO1-PTPN1, derived from a primary refractory Hodgkin's lymphoma. Both cell lines show equally high telomerase activity but U-HO1-PTPN differs from U-HO1 by a three times longer doubling time, low STAT5A expression, accumulation of RS-cells (p As expected, multinuclear U-HO1-RS-cells and multinuclear U-HO1-PTPN1-RS-cells differ from their mononuclear H-precursors by their nuclear volume (p Conclusion Abundant RS-cells without additional very short telomeres including "t-stumps", high rate of apoptosis, but low STAT5A expression, are hallmarks of the U-HO1-PTPN1 cell line. These characteristics are independent of telomerase activity. Thus, PTPN1 induced dephosphorylation of STAT5 with consecutive lack of Akt/PKB activation and cellular arrest in G2, promoting induction of apoptosis, appears as a possible pathogenetic mechanism deserving further experimental investigation.

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

LENUS (Irish Health Repository)

Stordal, Britta

2013-06-01

Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

Science.gov (United States)

Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia; O'Brien, Thomas

2016-01-01

Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

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Yang Xiao

Full Text Available Nicotinamide adenine dinucleotide (NAD is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM to nicotinamide mononucleotide (NMN, the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334, one that shows intermediate sensitivity (NCI-H441, and one that is insensitive (LC-KJ. Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP and had lower reactive oxygen species (ROS levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

Artificial neural networks for classification in metabolomic studies of whole cells using 1H nuclear magnetic resonance.

LENUS (Irish Health Repository)

Brougham, D F

2011-01-01

We report the successful classification, by artificial neural networks (ANNs), of (1)H NMR spectroscopic data recorded on whole-cell culture samples of four different lung carcinoma cell lines, which display different drug resistance patterns. The robustness of the approach was demonstrated by its ability to classify the cell line correctly in 100% of cases, despite the demonstrated presence of operator-induced sources of variation, and irrespective of which spectra are used for training and for validation. The study demonstrates the potential of ANN for lung carcinoma classification in realistic situations.

Investigation of the selenium metabolism in cancer cell lines

DEFF Research Database (Denmark)

Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

2011-01-01

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

Generation of iPSC lines from primary human chorionic villi cells

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Björn Lichtner

2015-11-01

Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

Pharmacophore-based screening of differentially-expressed PGF, DDIT4, COMP and CHI3L1 from hMSC cell lines reveals five novel therapeutic compounds for primary osteoporosis

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Catherine Jessica Lai

2016-06-01

Full Text Available As many societies age, primary osteoporosis (PO is increasingly a major health problem. Current drug treatments such as alendronate and risedronate have known side effects. We took an agnostic empirical approach to find PO therapeutic compounds. We examined 13,548,960 probe data-points from mesenchymal stromal cell (hMSC lines and found that PGF, DDIT4, and COMP to be up-regulated, and CHI3L1, down-regulated. We then identified their druggable domains. For the up-regulated differentially-expressed genes, we used protein–protein interactions to find residue clusters as binding surfaces. We then employed pharmacophore models to screen 15,407,096 conformations of 22,723,923 compounds, which identified (6R,9R-6-(2-furyl-9-(1H-indol-3-yl-2-(trifluoromethyl-5,6,7, 9-tetrahydro-4H[1,2,4]triazolo[5,1],(2S-N1-[2-[2-(methylamino-2-oxo-ethyl]phenyl]-N2-phenylpyrrolidine-1,2-dicarboxamide, and 2-furyl-(1H-indol-3-yl-methyl-BLAHone as candidate compounds. For the down-regulated CH13L1, we relied on genome-wide disease signatures to identify (11alpha-9-fluoro-11,17,21-trihydroxypregn-4-ene-3,20-dione and Genistein as candidate compounds. Our approach differs from previous research as we did not confine our drug targets to hypothesized compounds in the existing literature. Instead, we allowed the full expression profile of PO cell lines to reveal the most desirable targets. Second, our differential gene analysis revealed both up- and down-regulated genes, in contrast to the literature, which has focused on inhibiting only up-regulated genes. Third, our virtual screening universe of 22,723,923 compounds was more than 100 times larger than those in the known literature.

Effects of Monoclonal Antibody Cetuximab on Proliferation of Non-small Cell Lung Cancer Cell lines

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Zhen CHEN

2010-08-01

Full Text Available Background and objective The epidermal growth factor receptor (EGFR monoclonal antibody cetuximab has been used widely in non-small cell lung cancer patients. The aim of this study is to explore the effect of lung cancer cells (A549, H460, H1299, SPC-A-1 which were treated by cetuximab in vitro. Methods We studied the effects of increasing concentrations of cetuximab (1 nmol/L-625 nmol/L in four human lung cancer cell lines (A549, SPC-A-1, H460, H1229. CCK8 measured the inhibition of cell proliferation in each group. A549, SPC-A-1 were marked by PI and the statuses of apoptosis were observed. Western blot were used to detect the proliferation-related signaling protein and apoptosis-related protein in A549. Results The treatment with cetuximab resulted in the effect on cell proliferation and apoptosis in a time- and dosedependent manner. The expression of activated key enzymes (p-AKT, p-EGFR, p-MAPK in EGFR signaling transduction pathway were down-regulated more obviously. Conclusion Cetuximab is an effective targeted drug in the treatment of lung cancer cell lines, tissues, most likely to contribute to the inhibition of key enzymes in EGFR signaling transduction pathway.

Antitumor potential of 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H-pyrazoles in human bladder cancer cells.

Science.gov (United States)

Tessmann, Josiane Weber; Buss, Julieti; Begnini, Karine Rech; Berneira, Lucas Moraes; Paula, Favero Reisdorfer; de Pereira, Claudio Martin Pereira; Collares, Tiago; Seixas, Fabiana Kömmling

2017-10-01

Bladder cancer is a genitourinary malignant disease common worldwide. Current chemotherapy is often limited mainly due to toxicity and drug resistance. Thus, there is a continued need to discover new therapies. Recently evidences shows that pyrazoline derivatives are promising antitumor agents in many types of cancers, but there are no studies with bladder cancer. In order to find potent and novel chemotherapy drugs for bladder cancer, a series of pyrazoline derivatives 2a-2d were tested for their antitumor activity in two human bladder cancer cell lines 5647 and T24. The MTT assay showed that the compounds 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-1H-pyrazole (2a) and 1-thiocarbamoyl-5-(4-chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole (2c) decrease the cell viability of 5637 cells. Molecular modeling indicated that these compounds had a good oral bioavailability and low toxicities. Clonogenic assay and flow cytometric analysis were used to assess colony formation, apoptosis induction and cell cycle distribution. Overall, our results suggest that pyrazoline 2a and 2c, with the substituents hydrogen and chlorine respectively, may decrease cell viability and colony formation of bladder cancer 5637 cell line by inhibition of cell cycle progression, and for pyrazoline 2a, by induction of apoptosis. As indicated by the physicochemical properties of these compounds, the steric factor influences the activity. Therefore, these pyrazoline derivatives can be considered promising anticancer agents for the treatment of bladder cancer. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses.

Science.gov (United States)

Chiapponi, Chiara; Zanni, Irene; Garbarino, Chiara; Barigazzi, Giuseppe; Foni, Emanuela

2010-01-01

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, pH1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p<0.01).

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

Science.gov (United States)

Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

2013-08-01

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness.

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Yu-Fen Tseng

Full Text Available Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14, a reassortant virus between A/Vietnam/1194/2004 (H5N1 virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15 was generated and can grow over 10(8 TCID(50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy.

Science.gov (United States)

Wang, Wenjun; Wang, Qing; Wan, Danyang; Sun, Yue; Wang, Lin; Chen, Hong; Liu, Chengyu; Petersen, Robert B; Li, Jianshuang; Xue, Weili; Zheng, Ling; Huang, Kun

2017-05-04

Autophagy plays critical and complex roles in many human diseases, including diabetes and its complications. However, the role of autophagy in the development of diabetic retinopathy remains uncertain. Core histone modifications have been reported involved in the development of diabetic retinopathy, but little is known about the histone variants. Here, we observed increased autophagy and histone HIST1H1C/H1.2, an important variant of the linker histone H1, in the retinas of type 1 diabetic rodents. Overexpression of histone HIST1H1C upregulates SIRT1 and HDAC1 to maintain the deacetylation status of H4K16, leads to upregulation of ATG proteins, then promotes autophagy in cultured retinal cell line. Histone HIST1H1C overexpression also promotes inflammation and cell toxicity in vitro. Knockdown of histone HIST1H1C reduces both the basal and stresses (including high glucose)-induced autophagy, and inhibits high glucose induced inflammation and cell toxicity. Importantly, AAV-mediated histone HIST1H1C overexpression in the retinas leads to increased autophagy, inflammation, glial activation and neuron loss, similar to the pathological changes identified in the early stage of diabetic retinopathy. Furthermore, knockdown of histone Hist1h1c by siRNA in the retinas of diabetic mice significantly attenuated the diabetes-induced autophagy, inflammation, glial activation and neuron loss. These results indicate that histone HIST1H1C may offer a novel therapeutic target for preventing diabetic retinopathy.

Two distinct affinity binding sites for IL-1 on human cell lines

International Nuclear Information System (INIS)

Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

1989-01-01

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

Suberoylanilide hydroxamic acid affects γH2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

International Nuclear Information System (INIS)

Blattmann, C.; Oertel, S.; Thiemann, M.; Weber, K.J.; Schmezer, P.; Zelezny, O.; Lopez Perez, R.; Kulozik, A.E.; Debus, J.; Ehemann, V.

2012-01-01

Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by γH2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. γH2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, γH2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors. (orig.)

The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells.

Science.gov (United States)

Fortunato, Angelo

2017-08-01

The transition of cells from the epithelial to the mesenchymal state (EMT) plays an important role in tumor progression. EMT allows cells to acquire mobility, stem-like behavior and resistance to apoptosis and drug treatment. These features turn EMT into a central process in tumor biology. Ion channels are attractive targets for the treatment of cancer since they play critical roles in controlling a wide range of physiological processes that are frequently deregulated in cancer. Here, we investigated the role of ether-a-go-go-related 1 (hERG1) ion channels in the EMT of colorectal cancer cells. We studied the epithelial-mesenchymal profile of different colorectal cancer-derived cell lines and the expression of hERG1 potassium channels in these cell lines using real-time PCR. Next, we knocked down hERG1 expression in HCT116 cells using lentivirus mediated RNA interference and characterized the hERG1 silenced cells in vitro and in vivo. Finally, we investigated the capacity of riluzole, an ion channel-modulating drug used in humans to treat amyotrophic lateral sclerosis, to reduce the resistance of the respective colorectal cancer cells to the chemotherapeutic drug cisplatin. We found that of the colorectal cancer-derived cell lines tested, HCT116 showed the highest mesenchymal profile and a high hERG1 expression. Subsequent hERG1 expression knockdown induced a change in cell morphology, which was accompanied by a reduction in the proliferative and tumorigenic capacities of the cells. Notably, we found that hERG1expression knockdown elicited a reversion of the EMT profile in HCT116 cells with a reacquisition of the epithelial-like profile. We also found that riluzole increased the sensitivity of HCT116 cisplatin-resistant cells to cisplatin. Our data indicate that hERG1 plays a role in the EMT of colorectal cancer cells and that its knockdown reduces the proliferative and tumorigenic capacities of these cells. In addition, we conclude that riluzole may be used in

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Science.gov (United States)

Hamilton, Gerhard

2014-01-01

Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

Science.gov (United States)

Rütgen, Barbara C; Willenbrock, Saskia; Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Murua Escobar, Hugo

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Transcriptome variations among human embryonic stem cell lines are associated with their differentiation propensity.

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Changbin Sun

Full Text Available Human embryonic stem cells (hESCs have the potential to form any cell type in the body, making them attractive cell sources in drug screening, regenerative medicine, disease and developmental processes modeling. However, not all hESC lines have the equal potency to generate desired cell types in vitro. Significant variations have been observed for the differentiation efficiency of various human ESC lines. The precise underpinning molecular mechanisms are still unclear. In this work, we compared transcriptome variations of four hESC lines H7, HUES1, HUES8 and HUES9. We found that hESC lines have different gene expression profiles, and these differentially expressed genes (DEGs are significantly enriched in developmental processes, such as ectodermal, mesodermal and endodermal development. The enrichment difference between hESC lines was consistent with its lineage bias. Among these DEGs, some pluripotency factors and genes involved in signaling transduction showed great variations as well. The pleiotropic functions of these genes in controlling hESC identity and early lineage specification, implicated that different hESC lines may utilize distinct balance mechanisms to maintain pluripotent state. When the balance is broken in a certain environment, gene expression variation between them could impact on their different lineage specification behavior.

Establishment and characterization of a novel head and neck squamous cell carcinoma cell line USC-HN1.

Science.gov (United States)

Liebertz, Daniel J; Lechner, Melissa G; Masood, Rizwan; Sinha, Uttam K; Han, Jing; Puri, Raj K; Correa, Adrian J; Epstein, Alan L

2010-02-22

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and lethal malignancy. Publically available cell lines are mostly of lingual origin, or have not been carefully characterized. Detailed characterization of novel HNSCC cell lines is needed in order to provide researchers a concrete keystone on which to build their investigations. The USC-HN1 cell line was established from a primary maxillary HNSCC biopsy explant in tissue culture. The immortalized cells were then further characterized by heterotransplantation in Nude mice; immunohistochemical staining for relevant HNSCC biomarkers; flow cytometry for surface markers; cytogenetic karyotypic analysis; human papillomavirus and Epstein-Barr virus screening; qRT-PCR for oncogene and cytokine analysis; investigation of activated, cleaved Notch1 levels; and detailed 35,000 gene microarray analysis. Characterization experiments confirmed the human HNSCC origin of USC-HN1, including a phenotype similar to the original tumor. Viral screening revealed no HPV or EBV infection, while western blotting displayed significant upregulation of activated, cleaved Notch1. USC-HN1, a novel immortalized cell line has been derived from a maxillary HNSCC. Characterization studies have shown that the cell line is of HNSCC origin and displays many of the same markers previously reported in the literature. USC-HN1 is available for public research and will further the investigation of HNSCC and the development of new therapeutic modalities.

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1

DEFF Research Database (Denmark)

Abdallah, Basem M; Jensen, Charlotte H; Gutierrez, Gloria

2004-01-01

Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION......: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have...... was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high...

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

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Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

2010-09-01

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay. Copyright 2010 Elsevier Ltd. All rights reserved.

The peculiar radio-loud narrow line Seyfert 1 galaxy 1H 0323+342

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Paliya, Vaidehi S.; Stalin, C. S. [Indian Institute of Astrophysics, Block-II, Koramangala, Bangalore-560034 (India); Sahayanathan, S. [Astrophysical Science Division, Bhabha Atomic Research Center, Mumbai-400085 (India); Parker, M. L.; Fabian, A. C. [Institute of Astronomy, Madingley Road, Cambridge CB3 0HA (United Kingdom); Anjum, Ayesha [Department of Physics, Christ University, Bangalore-560029 (India); Pandey, S. B., E-mail: vaidehi@iiap.res.in [Aryabhatta Research Institute of Observational Sciences, Manora peak, Nainital-263129 (India)

2014-07-10

We present a multiwavelength study of the radio-loud narrow-line Seyfert 1 galaxy (NLSy1) 1H 0323+342, detected by the Fermi Gamma-Ray Space Telescope. Multiband light curves show many orphan X-ray and optical flares having no corresponding γ-ray counterparts. Such anomalous variability behavior can be due to different locations of the emission region from the central source. During a large flare, a γ-ray flux doubling timescale as small as ∼3 hr is noticed. We built spectral energy distributions (SEDs) during different activity states and modeled them using a one-zone leptonic model. The shape of the optical/UV component of the SEDs is dominated by accretion disk emission in all the activity states. In the X-ray band, significant thermal emission from the hot corona is inferred during quiescent and first flaring states; however, during subsequent flares, the nonthermal jet component dominates. The γ-ray emission in all the states can be well explained by inverse-Compton scattering of accretion disk photons reprocessed by the broad-line region. The source showed violent intra-night optical variability, coinciding with one of the high γ-ray activity states. An analysis of the overall X-ray spectrum fitted with an absorbed power-law plus relativistic reflection component hints at the presence of an Fe Kα line and returns a high black hole spin value of a = 0.96 ± 0.14. We argue that 1H 0323+342 possesses dual characteristics, akin to both flat-spectrum radio quasars (FSRQs) and radio-quiet NLSy1 galaxies, though at a low jet power regime compared to powerful FSRQs.

Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.

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Strzelecka-Kiliszek, Agnieszka; Bozycki, Lukasz; Mebarek, Saida; Buchet, Rene; Pikula, Slawomir

2017-06-01

Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level. Copyright © 2017 Elsevier Inc. All rights reserved.

Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1 viruses.

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Marion Desdouits

Full Text Available Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1 in 2009. The pathogenesis of these influenza-associated myopathies (IAM remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1 isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes were highly susceptible to infection by both influenza A(H1N1 isolates, whereas undifferentiated cells (i. e. myoblasts were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

Effects of irradiation on the expression of the adhesion molecules (NCAM, ICAM-1) by glioma cell lines

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Yamanaka, Ryuya; Tanaka, Ryuichi; Yoshida, Seiichi [Niigata Univ. (Japan). Brain Research Inst.

1993-11-01

The expression of the intercellular adhesion molecule-1 (ICAM-1) and neural cell adhesion molecule (NCAM) by glioma cell lines was investigated. The effects of interferon (IFN)-[gamma] or irradiation on the expression was also assessed. Two glioma cell lines showed more than 75% NCAM-positive cells. After treatment with IFN-[gamma] or irradiation, another three cell lines were induced to show more than 50% positive cells. Three glioma cell lines showed more than 50% ICAM-1-positive cells. After treatment with IFN-[gamma], another two cell lines were induced to show more than 50% positive cells. After treatment with irradiation, one more cell line was induced to show more than 50% positive cells. ICAM-1 and NCAM expression by glioma cell lines is susceptible to modulation by IFN-[gamma] or irradiation. (author).

Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood–brain barrier in rats

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Valiante S

2015-03-01

Full Text Available Salvatore Valiante,1,* Annarita Falanga,2,3,* Luisa Cigliano,1 Giuseppina Iachetta,1 Rosa Anna Busiello,1 Valeria La Marca,1 Massimiliano Galdiero,4 Assunta Lombardi,1 Stefania Galdiero1,2 1Department of Biology, 2Department of Pharmacy, 3DFM Scarl, University of Naples Federico II, 4Department of Experimental Medicine, II University of Naples, Naples, Italy *These authors contributed equally to this paper and are considered joint first authors Abstract: Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood–brain barrier (BBB. In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases. Keywords: drug delivery, neurons, astrocytes, blood–brain barrier, peptide

Functional importance of GLP-1 receptor species and expression levels in cell lines.

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Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

2012-04-10

Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

Time-Qualified Patterns of Variation of PPARγ, DNMT1, and DNMT3B Expression in Pancreatic Cancer Cell Lines

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Valerio Pazienza

2012-01-01

Full Text Available Carcinogenesis is related to the loss of homeostatic control of cellular processes regulated by transcriptional circuits and epigenetic mechanisms. Among these, the activities of peroxisome proliferator-activated receptors (PPARs and DNA methyltransferases (DNMTs are crucial and intertwined. PPARγ is a key regulator of cell fate, linking nutrient sensing to transcription processes, and its expression oscillates with circadian rhythmicity. Aim of our study was to assess the periodicity of PPARγ and DNMTs in pancreatic cancer (PC. We investigated the time-related patterns of PPARG, DNMT1, and DNMT3B expression monitoring their mRNA levels by qRT-PCR at different time points over a 28-hour span in BxPC-3, CFPAC-1, PANC-1, and MIAPaCa-2 PC cells after synchronization with serum shock. PPARG and DNMT1 expression in PANC-1 cells and PPARG expression in MIAPaCa-2 cells were characterized by a 24 h period oscillation, and a borderline significant rhythm was observed for the PPARG, DNMT1, and DNMT3B expression profiles in the other cell lines. The time-qualified profiles of gene expression showed different shapes and phase relationships in the PC cell lines examined. In conclusion, PPARG and DNMTs expression is characterized by different time-qualified patterns in cell lines derived from human PC, and this heterogeneity could influence cell phenotype and human disease behaviour.

The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells.

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Jeffery, N; Richardson, S; Beall, C; Harries, L W

2017-12-15

Interaction between islet cell subtypes and the extracellular matrix influences beta-cell function in mammals. The tissue architecture of rodent islets is very different to that of human islets; cell-to-cell communication and interaction with the extracellular matrix may vary between species. In this work, we have compared the responses of the human EndoC-βH1 cell line to non-human and human-derived growth matrices in terms of growth morphology, gene expression and glucose-stimulated insulin secretion (GSIS). EndoC-βH1 cells demonstrated a greater tendency to form cell clusters when cultured in a human microenvironment and exhibited reduced alpha cell markers at the mRNA level; mean expression difference - 0.23 and - 0.51; p = 0.009 and 0.002 for the Aristaless-related homeobox (ARX) and Glucagon (GCG) genes respectively. No differences were noted in the protein expression of mature beta cell markers such as Pdx1 and NeuroD1 were noted in EndoC-βH1 cells grown in a human microenvironment but cells were however more sensitive to glucose (4.3-fold increase in insulin secretion following glucose challenge compared with a 1.9-fold increase in cells grown in a non-human microenvironment; p = 0.0003). Our data suggests that the tissue origin of the cellular microenvironment has effects on the function of EndoC-βH1 cells in vitro, and the use of a more human-like culture microenvironment may bring benefits in terms of increased physiological relevance. Copyright © 2017 Elsevier Inc. All rights reserved.

Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD for monogenic disorder

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Abdelkrim Hmadcha

2016-05-01

Full Text Available From 106 human blastocyts donate for research after in vitro fertilization (IVF and preimplantation genetic diagnosis (PGD for monogenetic disorder, 3 human embryonic stem cells (hESCs HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15(q34.3;q14 detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

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Barbara C Rütgen

Full Text Available Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL. Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-γ(c (-/- mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+, MHCII(+, CD11a(+ and CD79αcy(+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

International Nuclear Information System (INIS)

Youakim, A.; Herscovics, A.

1985-01-01

The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2- 3 H]mannose or L-[5,6- 3 H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2- 3 H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2- 3 H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6- 3 H]glucosamine and L-[1- 14 C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3 H-labeled N-acetylglucosamine and N-acetylgalactosamine

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia.

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Fernández Calotti, Paula; Galmarini, Carlos María; Cañones, Cristian; Gamberale, Romina; Saénz, Daniel; Avalos, Julio Sánchez; Chianelli, Mónica; Rosenstein, Ruth; Giordano, Mirta

2008-02-15

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20 ng/ml for 24 h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Significance of the neurotensin receptor Na+/H+-exchanger 1 axis in human pancreatic cancer cells

International Nuclear Information System (INIS)

Olszewski, U.

2009-01-01

Pancreatic cancer is characterized by early dissemination and rapid acquisition of drug resistance, resulting in dismal prognosis in patients. New targeted therapies failed to improve the low five-year survival rates. Characterization of neuropeptides as growth factors for pancreatic cancer cells stimulated interest in the development of suitable inhibitors. In particular, neurotensin (NT) stimulated proliferation of cancer cell lines, and the NT receptor 1 (NTR1) antagonist SR48692 was found to inhibit growth of tumor xenografts. However, clinical application of SR48692 in small cell lung cancer failed to yield significant responses. Nevertheless, expression of NTRs in more than 90% of pancreatic tumors points to an important role of the NT - NTR system in this tumor entity. Therefore, the present study aimed at investigation of the significance of NT - NTR signaling by use of BxPC-3, PANC-1 and MIA PaCa-2 pancreatic cancer cells and the NTR-positive HT-29 colon carcinoma cell line for comparison. Functional NTR1 that triggers release of intracellular Ca 2+ upon binding of the stable NT analog Lys 8 -Ψ-Lys 9 NT(8-13) was confirmed in all pancreatic cancer cell lines. The fraction of cells in S phase was increased in response to the NT analog and proliferation of the pancreatic cancer cells stimulated to a limited extent. In contrast to epidermal growth factor receptor (EGFR), NTR1 expression was found to reach a maximum in confluent cultures of resting (G1/0 phase) BxPC-3 and PANC-1 cells. In addition, again unlike EGFR, expression of NTR1 proved to be dependent on extracellular pH with highest levels under acidic conditions. Accordingly, Lys 8 -Ψ-Lys 9 NT(8-13) induced marked intracellular alkalinization in BxPC-3, PANC-1 and a panel of colon cancer cell lines and slight acidification in MIA PaCa-2 cells under conditions that confine regulation of intracellular pH to the ubiquitously expressed Na + /H + exchanger 1 (NHE1). Similar results were obtained in

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

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Gerhard Hamilton

2014-03-01

Full Text Available Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4 inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose-Polymerase 1 (PARP1 inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS.

Lymphoblast-derived integration-free iPSC line AD-TREM2-1 from a 67 year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant

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Soraia Martins

2018-05-01

Full Text Available Human lymphoblast cells from a male diagnosed with Alzheimer's disease (AD expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem cells (iPSCs by over-expressing episomal-based plasmids harbouring OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. AD-TREM2–1 was defined as pluripotent based on (i expression of pluripotency-associated markers (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.947.

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

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Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

2014-02-01

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

International Nuclear Information System (INIS)

Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S.; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia

2013-01-01

A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

Energy Technology Data Exchange (ETDEWEB)

Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S., E-mail: ccelso@dcm.ufrpe.br [Universidade Federal Rural de Pernambuco (LSCB/UFRPE), Recife, PE (Brazil). Dept. de Ciencias Moleculares. Lab. de Sintese de Compostos Bioativos; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia [Universidade Federal do Ceara (LOE/UFC), Fortaleza, CE (Brazil). Dept. de Fisiologia e Farmacologia. Lab. de Oncologia Experimental

2013-09-15

A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA).

Science.gov (United States)

Ashikaga, Takao; Sakaguchi, Hitoshi; Sono, Sakiko; Kosaka, Nanae; Ishikawa, Makie; Nukada, Yuko; Miyazawa, Masaaki; Ito, Yuichi; Nishiyama, Naohiro; Itagaki, Hiroshi

2010-08-01

We previously developed the human cell-line activation test (h-CLAT) in vitro skin sensitisation test, based on our reported finding that a 24-hour exposure of THP-1 cells (a human monocytic leukaemia cell line) to sensitisers is sufficient to induce the augmented expression of CD86 and CD54. The aim of this study is to confirm the predictive value of h-CLAT for skin sensitisation activity by employing a larger number of test chemicals. One hundred chemicals were selected, according to their categorisation in the local lymph node assay (LLNA), as being: extreme, strong, moderate and weak sensitisers, and non-sensitisers. The correlation of the h-CLAT results with the LLNA results was 84%. There were some false negatives (e.g. benzoyl peroxide, hexyl cinnamic aldehyde) and some false positives (e.g. 1-bromobutane, diethylphthalate). Eight out of the 9 false negatives (89%) were water-insoluble chemicals. The h-CLAT could positively predict not only extreme and strong sensitisers, but also moderate and weak sensitisers, though the detection rates of weak sensitisers and non-sensitisers were comparatively low. Some sensitisers enhanced both CD86 and CD54 levels, and some enhanced the level of only one of them. The use of the combination of CD86 and CD54 induction as a positive indicator, improved the accuracy of the test. In conclusion, the h-CLAT is expected to be a useful cell-based in vitro method for predicting skin sensitisation potential. 2010 FRAME.

Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration

International Nuclear Information System (INIS)

Guelden, Michael; Moerchel, Sabine; Seibert, Hasso

2005-01-01

Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC 50 values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC 50 values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC 50 values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity

LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

Science.gov (United States)

Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

2017-11-28

Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

Derivation from an alloreactive T-cell line of a clone which cross-reacts with a self H2-E-restricted minor alloantigen

DEFF Research Database (Denmark)

Owens, T; Liddell, M E; Crispe, I N

1984-01-01

An alloreactive T-helper-cell line [(A.TH X Balb/c) anti-A.TL] was shown to recognize both H2-Ek and H2-Ed. Both proliferation and polyclonal B-cell activation (protein A plaques) were used in the analyses of specificity. On cloning, the H2-Ek/Ed cross-reaction was shown by one clonotype...

Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder

OpenAIRE

Abdelkrim Hmadcha; Yolanda Aguilera; Maria Dolores Lozano-Arana; Nuria Mellado; Javier Sánchez; Cristina Moya; Luis Sánchez-Palazón; Jose Palacios; Guillermo Antiñolo; Bernat Soria

2016-01-01

From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were r...

Release of volatile organic compounds (VOCs from the lung cancer cell line CALU-1 in vitro

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Schubert Jochen

2008-11-01

Full Text Available Abstract Background The aim of this work was to confirm the existence of volatile organic compounds (VOCs specifically released or consumed by lung cancer cells. Methods 50 million cells of the human non-small cell lung cancer (NSCLC cell line CALU-1 were incubated in a sealed fermenter for 4 h or over night (18 hours. Then air samples from the headspace of the culture vessel were collected and preconcentrated by adsorption on solid sorbents with subsequent thermodesorption and analysis by means of gas chromatography mass spectrometry (GC-MS. Identification of altogether 60 compounds in GCMS measurement was done not only by spectral library match, but also by determination of retention times established with calibration mixtures of the respective pure compounds. Results The results showed a significant increase in the concentrations of 2,3,3-trimethylpentane, 2,3,5-trimethylhexane, 2,4-dimethylheptane and 4-methyloctane in the headspace of CALU-1 cell culture as compared to medium controls after 18 h. Decreased concentrations after 18 h of incubation were found for acetaldehyde, 3-methylbutanal, butyl acetate, acetonitrile, acrolein, methacrolein, 2-methylpropanal, 2-butanone, 2-methoxy-2-methylpropane, 2-ethoxy-2-methylpropane, and hexanal. Conclusion Our findings demonstrate that certain volatile compounds can be cancer-cell derived and thus indicative of the presence of a tumor, whereas other compounds are not released but seem to be consumed by CALU-1 cells.

Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

International Nuclear Information System (INIS)

Maroschik, Belinda; Gürtler, Anne; Krämer, Anne; Rößler, Ute; Gomolka, Maria; Hornhardt, Sabine; Mörtl, Simone; Friedl, Anna A

2014-01-01

Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

DEFF Research Database (Denmark)

Xu, Xun; Pan, Shengkai; Liu, Xin

2011-01-01

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

Photoperiod-H1 (Ppd-H1) Controls Leaf Size1[OPEN

Science.gov (United States)

Digel, Benedikt; Tavakol, Elahe; Verderio, Gabriele; Xu, Xin

2016-01-01

Leaf size is a major determinant of plant photosynthetic activity and biomass; however, it is poorly understood how leaf size is genetically controlled in cereal crop plants like barley (Hordeum vulgare). We conducted a genome-wide association scan for flowering time, leaf width, and leaf length in a diverse panel of European winter cultivars grown in the field and genotyped with a single-nucleotide polymorphism array. The genome-wide association scan identified PHOTOPERIOD-H1 (Ppd-H1) as a candidate gene underlying the major quantitative trait loci for flowering time and leaf size in the barley population. Microscopic phenotyping of three independent introgression lines confirmed the effect of Ppd-H1 on leaf size. Differences in the duration of leaf growth and consequent variation in leaf cell number were responsible for the leaf size differences between the Ppd-H1 variants. The Ppd-H1-dependent induction of the BARLEY MADS BOX genes BM3 and BM8 in the leaf correlated with reductions in leaf size and leaf number. Our results indicate that leaf size is controlled by the Ppd-H1- and photoperiod-dependent progression of plant development. The coordination of leaf growth with flowering may be part of a reproductive strategy to optimize resource allocation to the developing inflorescences and seeds. PMID:27457126

Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

International Nuclear Information System (INIS)

Yang, Qiwei; Tian, Yufeng; Ostler, Kelly R; Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Godley, Lucy A; Cohn, Susan L

2010-01-01

Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

Type I and type II interferons upregulate functional type I interleukin-1 receptor in a human fibroblast cell line TIG-1.

Science.gov (United States)

Takii, T; Niki, N; Yang, D; Kimura, H; Ito, A; Hayashi, H; Onozaki, K

1995-12-01

The regulation of type I interleukin-1 receptor (IL-1R) expression by type I, interferon (IFN)-alpha A/D, and type II IFN, IFN-gamma, in a human fibroblast cell line TIG-1 was investigated. After 2 h stimulation with human IFN-alpha A/D or IFN-gamma, the levels of type I IL-1R mRNA increased. We previously reported that IL-1 upregulates transcription and cell surface molecules of type I IL-1R in TIG-1 cells through induction of prostaglandin (PG) E2 and cAMP accumulation. However, indomethacin was unable to inhibit the effect of IFNs, indicating that IFNs augment IL-1R expression through a pathway distinct from that of IL-1. The augmentation was also observed in other fibroblast cell lines. Nuclear run-on assays and studies of the stability of mRNA suggested that the increase in IL-1R mRNA was a result of the enhanced transcription of IL-1R gene. Binding studies using 125I-IL-1 alpha revealed that the number of cell surface IL-1R increased with no change in binding affinity by treatment with these IFNs. Pretreatment of the cells with IFNs enhanced IL-1-induced IL-6 production, indicating that IFNs upregulate functional IL-1R. IL-1 and IFNs are produced by the same cell types, as well as by the adjacent different cell types, and are concomitantly present in lesions of immune and inflammatory reactions. These results therefore suggest that IFNs exhibit synergistic effects with IL-1 through upregulation of IL-1R. Augmented production of IL-6 may also contribute to the reactions.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

Energy Technology Data Exchange (ETDEWEB)

Thanan, Raynoo [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Techasen, Anchalee [Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Faculty of Associated Medical Science, Khon Kaen University, Khon Kaen 40002 (Thailand); Hou, Bo [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507 (Japan); Jamnongkan, Wassana; Armartmuntree, Napat [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Yongvanit, Puangrat, E-mail: puangrat@kku.ac.th [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Murata, Mariko, E-mail: mmurata@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507 (Japan)

2015-08-14

Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H{sub 2}O{sub 2}) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H{sub 2}O{sub 2}-resistant cell line from an immortal cholangiocyte cell line (MMNK1) by chronic treatment with low-concentration H{sub 2}O{sub 2} (25 μM). After 72 days of induction, H{sub 2}O{sub 2}-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H{sub 2}O{sub 2}-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H{sub 2}O{sub 2}). These findings suggest that H{sub 2}O{sub 2}-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes. - Highlights: • An H{sub 2}O{sub 2}-resistant ox-MMNK1-L cells was established from

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo.

Science.gov (United States)

Yang, Xueling; Li, Xiaoli; Guo, Zhi; Si, Tongguo; Yu, Haipeng; Xing, Wenge

2018-02-01

To describe immunological consequences induced by cryoablation against H22 cells in vivo. Adult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected. Compared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21. Our study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell. Copyright © 2017. Published by Elsevier Inc.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

Science.gov (United States)

Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

2012-10-01

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

Clostridium difficile toxin B inhibits the secretory response of human mast cell line-1 (HMC-1) cells stimulated with high free-Ca2+ and GTPγS

International Nuclear Information System (INIS)

Balletta, Andrea; Lorenz, Dorothea; Rummel, Andreas; Gerhard, Ralf; Bigalke, Hans; Wegner, Florian

2015-01-01

Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca 2+ and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (C m ) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell “stimulated” C m increase, as well as on “constitutive” secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3–24 h exhibited a significant reduction of the mean absolute and relative C m increase in response to free-Ca 2+ and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity

Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

Science.gov (United States)

Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers. Copyright 2009 Elsevier Inc. All rights reserved.

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

International Nuclear Information System (INIS)

Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

2010-01-01

Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

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Kellermeier Silvia

2010-06-01

Full Text Available Abstract Background Cholangiocarcinoma (CC is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Methods Expression of EGFR (epithelial growth factor receptor, HGFR (hepatocyte growth factor receptor IGF1R (insulin-like growth factor 1 receptor, IGF2R (insulin-like growth factor 2 receptor and VEGFR1-3 (vascular endothelial growth factor receptor 1-3 were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1. The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. Results EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml, with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D. HuH28, OZ and TFK-1 lacked KRAS mutation. Conclusion CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

2015-04-03

Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

Cell lines derived from a Medaka radiation-sensitive mutant have defects in DNA double-strand break responses

International Nuclear Information System (INIS)

Hidaka, Masayuki; Oda, Shoji; Mitani, Hiroshi; Kuwahara, Yoshikazu; Fukumoto, Manabu

2010-01-01

It was reported that the radiation-sensitive Medaka mutant 'ric1' has a defect in the repair of DNA double-strand breaks (DSBs) induced by γ-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to γ-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after γ-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (γH2AX) foci were formed after γ-irradiation, however, the γH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of γH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro. (author)

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

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Stanislav V. Sosnovtsev

2017-02-01

Full Text Available The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1, was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

Photoperiod-H1 (Ppd-H1) Controls Leaf Size.

Science.gov (United States)

Digel, Benedikt; Tavakol, Elahe; Verderio, Gabriele; Tondelli, Alessandro; Xu, Xin; Cattivelli, Luigi; Rossini, Laura; von Korff, Maria

2016-09-01

Leaf size is a major determinant of plant photosynthetic activity and biomass; however, it is poorly understood how leaf size is genetically controlled in cereal crop plants like barley (Hordeum vulgare). We conducted a genome-wide association scan for flowering time, leaf width, and leaf length in a diverse panel of European winter cultivars grown in the field and genotyped with a single-nucleotide polymorphism array. The genome-wide association scan identified PHOTOPERIOD-H1 (Ppd-H1) as a candidate gene underlying the major quantitative trait loci for flowering time and leaf size in the barley population. Microscopic phenotyping of three independent introgression lines confirmed the effect of Ppd-H1 on leaf size. Differences in the duration of leaf growth and consequent variation in leaf cell number were responsible for the leaf size differences between the Ppd-H1 variants. The Ppd-H1-dependent induction of the BARLEY MADS BOX genes BM3 and BM8 in the leaf correlated with reductions in leaf size and leaf number. Our results indicate that leaf size is controlled by the Ppd-H1- and photoperiod-dependent progression of plant development. The coordination of leaf growth with flowering may be part of a reproductive strategy to optimize resource allocation to the developing inflorescences and seeds. © 2016 American Society of Plant Biologists. All rights reserved.

Synthesis and Preliminary Cytotoxicity Studies of 1-[1-(4,5-Dihydrooxazol- 2-yl)-1H-indazol-3-yl]-3-phenylurea and 3-phenylthiourea Derivatives.

Science.gov (United States)

Kornicka, Anita; Saczewski, Franciszek; Bednarski, Patrick J; Korcz, Martyna; Szumlas, Piotr; Romejko, Ewa; Sakowicz, Aneta; Sitek, Lukasz; Wojciechowska, Monika

2017-01-01

N-substituted 3-amino-1H-indazoles represent an interesting class of biologically active compounds. Among them, derivatives containing phenylurea moiety are of particular interest. Such compounds have been found to possess inhibitory activity against cancer cell growth. Additionally, various oxazoline-containing compounds have also been designed as potential anticancer agents. The aim of this work was to obtain a new class of N-substituted 3-amino-1H-indazole derivatives with cytotoxic activity towards cancer cells. Two series of 1-[1-(4,5-dihydrooxazol-2-yl)-1H-indazol-3-yl]-3-phenylurea and 3- phenylthiourea derivatives 7-17 and 18-22, respectively, were prepared and screened for their potential in vitro cytotoxic activities against lung carcinoma LCLC-103H cell line using a crystal violet microtiter plate assay. All the urea derivatives, except the compound 8, were inactive at a concentration of 20 μM attainable in cancer cells, while the thiourea derivatives showed a pronounced cancer cell growth inhibitory effects. The most potent 1-[1-(4,5-dihydrooxazol-2-yl)-1H-indazol-3-yl]-3-ptolylthiourea (19) exhibited cytotoxicity on the lung cancer LCLC-103H and cervical cancer SISO cell lines at a concentration of 10 µM. Moreover, compound 19 displayed cytostatic activity against pancreas cancer DAN-G cell line. The 1-[1-(4,5-dihydrooxazol-2-yl)-1H-indazol-3-yl]-3-phenylthiourea derivatives described herein may serve as a useful scaffold for the search for novel anticancer agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Derivation of NEM2 affected human embryonic stem cell line Genea079

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Biljana Dumevska

2016-03-01

Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells.

Science.gov (United States)

Gunthorpe, M J; Smith, G D; Davis, J B; Randall, A D

2001-08-01

Acid-sensing ion channels (ASICs) are a new and expanding family of proton-gated cation (Na+/Ca2+) channels that are widely expressed in sensory neurons and the central nervous system. Their distribution suggests that they may play a critical role in the sensation of the pain that accompanies tissue acidosis and may also be important in detecting the subtle pH variations that occur during neuronal signalling. Here, using whole-cell patch-clamp electrophysiology and reverse transcriptase-polymerase chain reaction (RT-PCR), we show that HEK293 cells, a commonly used cell line for the expression and characterisation of many ion channels, functionally express an endogenous proton-gated conductance attributable to the activity of human ASIC1a. These data therefore represent the first functional characterisation of hASIC1 and have many important implications for the use of HEK293 cells as a host cell system for the study of ASICs, vanilloid receptor-1 and any other proton-gated channel. With this latter point in mind we have devised a simple desensitisation strategy to selectively remove the contribution of hASIC1a from proton-gated currents recorded from HEK293 cells expressing vanilloid receptor-1.

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

DEFF Research Database (Denmark)

Xu, Xun; Pan, Shengkai; Liu, Xin

2011-01-01

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most....... Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance...... property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production....

Dynamic behavior of histone H1 microinjected into HeLa cells

International Nuclear Information System (INIS)

Wu, L.H.; Kuehl, L.; Rechsteiner, M.

1986-01-01

Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125 I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that of endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of ∼100h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts < 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

Science.gov (United States)

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

Science.gov (United States)

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

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Biljana Dumevska

2016-03-01

Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways

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Tang, Dongmei; Lin, Qin; He, Yingzi; Chai, Renjie; Li, Huawei

2016-01-01

The activation of neuromast (NM) supporting cell (SC) proliferation leads to hair cell (HC) regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of NM cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the NMs of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration. PMID:27303264

Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways

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Dongmei eTang

2016-05-01

Full Text Available The activation of neuromast supporting cell (SC proliferation leads to hair cell (HC regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of neuromast cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the neuromasts of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration.

Generation of OCIAD1 inducible overexpression human embryonic stem cell line: BJNhem20-OCIAD1-Tet-On

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Deeti K. Shetty

2016-03-01

Full Text Available Human embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010.

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells.

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Nishiyama, Takahito; Izawa, Tadashi; Usami, Mami; Ohnuma, Tomokazu; Ogura, Kenichiro; Hiratsuka, Akira

2010-04-09

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process. Copyright 2009 Elsevier Inc. All rights reserved.

Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line

DEFF Research Database (Denmark)

Long, M.; Laier, Peter; Vinggaard, Anne

2003-01-01

TV101L hepatoma cell lines. In comparison the results indicated that the rat H4IIE cell line is more sensitive than the human TV101L for detection of TCDD inducing AhR-CALUX activity. The pesticides iprodione, chlorpyrifos and prochloraz showed dose-dependent AhR agonistic effects in both cell lines...

Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone).

Science.gov (United States)

Isaka, Keiichi; Fujito, Atsuya; Sagawa, Yasukazu; Yudate, Tamaki; Nishi, Hirotaka; Ito, Hiroe; Takayama, Masaomi

2002-01-01

Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c-Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma.

MAML1 regulates cell viability via the NF-κB pathway in cervical cancer cell lines

International Nuclear Information System (INIS)

Kuncharin, Yanin; Sangphech, Naunpun; Kueanjinda, Patipark; Bhattarakosol, Parvapan; Palaga, Tanapat

2011-01-01

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and β-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-κB pathway was investigated, CaSki cells overexpressing DN

MAML1 regulates cell viability via the NF-{kappa}B pathway in cervical cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Kuncharin, Yanin [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Sangphech, Naunpun [Biotechnology Program, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Kueanjinda, Patipark [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Bhattarakosol, Parvapan [Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand); Palaga, Tanapat, E-mail: tanapat.p@chula.ac.th [Department of Microbiology, Faculty of Science, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330 (Thailand)

2011-08-01

The Notch signaling pathway plays important roles in tumorigenesis in a context-dependent manner. In human cervical cancer, alterations in Notch signaling have been reported, and both tumor-suppressing and tumor-promoting roles of Notch signaling have been proposed; however, the precise molecular mechanisms governing these roles in cervical cancer remain controversial. MAML is a transcriptional co-activator originally identified by its role in Notch signaling. Recent evidence suggests that it also plays a role in other signaling pathways, such as the p53 and {beta}-catenin pathways. MAML is required for stable formation of Notch transcriptional complexes at the promoters of Notch target genes. Chromosomal translocations affecting MAML have been shown to promote tumorigenesis. In this study, we used a truncated dominant-negative MAML1 (DN-MAML) to investigate the role of MAML in HPV-positive cervical cancer cell lines. Three human cervical cancer cell lines (HeLa, SiHa and CaSki) expressed all Notch receptors and the Notch target genes Hes1 and MAML1. Among these 3 cell lines, constitutive appearance of cleaved Notch1 was found only in CaSki cells, which suggests that Notch1 is constitutively activated in this cell line. Gamma secretase inhibitor (GSI) treatment, which suppresses Notch receptor activation, completely abrogated this form of Notch1 but had no effect on cell viability. Overexpression of DN-MAML by retroviral transduction in CaSki cells resulted in significant decreases in the mRNA levels of Hes1 and Notch1 but had no effects on the levels of MAML1, p53 or HPV E6/E7. DN-MAML expression induced increased viability of CaSki cells without any effect on cell cycle progression or cell proliferation. In addition, clonogenic assay experiments revealed that overexpression of DN-MAML resulted in increased colony formation compared to the overexpression of the control vector. When the status of the NF-{kappa}B pathway was investigated, CaSki cells overexpressing

What the public was saying about the H1N1 vaccine: perceptions and issues discussed in on-line comments during the 2009 H1N1 pandemic.

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Natalie Henrich

Full Text Available During the 2009 H1N1 pandemic, a vaccine was made available to all Canadians. Despite efforts to promote vaccination, the public's intent to vaccinate remained low. In order to better understand the public's resistance to getting vaccinated, this study addressed factors that influenced the public's decision making about uptake. To do this, we used a relatively novel source of qualitative data--comments posted on-line in response to news articles on a particular topic. This study analysed 1,796 comments posted in response to 12 articles dealing with H1N1 vaccine on websites of three major Canadian news sources. Articles were selected based on topic and number of comments. A second objective was to assess the extent to which on-line comments can be used as a reliable data source to capture public attitudes during a health crisis. The following seven themes were mentioned in at least 5% of the comments (% indicates the percentage of comments that included the theme: fear of H1N1 (18.8%; responsibility of media (17.8%; government competency (17.7%; government trustworthiness (10.7%; fear of H1N1 vaccine (8.1%; pharmaceutical companies (7.6%; and personal protective measures (5.8%. It is assumed that the more frequently a theme was mentioned, the more that theme influenced decision making about vaccination. These key themes for the public were often not aligned with the issues and information officials perceived, and conveyed, as relevant in the decision making process. The main themes from the comments were consistent with results from surveys and focus groups addressing similar issues, which suggest that on-line comments do provide a reliable source of qualitative data on attitudes and perceptions of issues that emerge in a health crisis. The insights derived from the comments can contribute to improved communication and policy decisions about vaccination in health crises that incorporate the public's views.

What the public was saying about the H1N1 vaccine: perceptions and issues discussed in on-line comments during the 2009 H1N1 pandemic.

Science.gov (United States)

Henrich, Natalie; Holmes, Bev

2011-04-18

During the 2009 H1N1 pandemic, a vaccine was made available to all Canadians. Despite efforts to promote vaccination, the public's intent to vaccinate remained low. In order to better understand the public's resistance to getting vaccinated, this study addressed factors that influenced the public's decision making about uptake. To do this, we used a relatively novel source of qualitative data--comments posted on-line in response to news articles on a particular topic. This study analysed 1,796 comments posted in response to 12 articles dealing with H1N1 vaccine on websites of three major Canadian news sources. Articles were selected based on topic and number of comments. A second objective was to assess the extent to which on-line comments can be used as a reliable data source to capture public attitudes during a health crisis. The following seven themes were mentioned in at least 5% of the comments (% indicates the percentage of comments that included the theme): fear of H1N1 (18.8%); responsibility of media (17.8%); government competency (17.7%); government trustworthiness (10.7%); fear of H1N1 vaccine (8.1%); pharmaceutical companies (7.6%); and personal protective measures (5.8%). It is assumed that the more frequently a theme was mentioned, the more that theme influenced decision making about vaccination. These key themes for the public were often not aligned with the issues and information officials perceived, and conveyed, as relevant in the decision making process. The main themes from the comments were consistent with results from surveys and focus groups addressing similar issues, which suggest that on-line comments do provide a reliable source of qualitative data on attitudes and perceptions of issues that emerge in a health crisis. The insights derived from the comments can contribute to improved communication and policy decisions about vaccination in health crises that incorporate the public's views.

Caveolin-1 influences human influenza A virus (H1N1 multiplication in cell culture

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Hemgård Gun-Viol

2010-05-01

Full Text Available Abstract Background The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. Results Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1 as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1 strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1 virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK, a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. Conclusion As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.

Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

Science.gov (United States)

Suijker, Johnny; Oosting, Jan; Koornneef, Annemarie; Struys, Eduard A; Salomons, Gajja S; Schaap, Frank G; Waaijer, Cathelijn J F; Wijers-Koster, Pauline M; Briaire-de Bruijn, Inge H; Haazen, Lizette; Riester, Scott M; Dudakovic, Amel; Danen, Erik; Cleton-Jansen, Anne-Marie; van Wijnen, Andre J; Bovée, Judith V M G

2015-05-20

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

Sulforaphane causes epigenetic repression of hTERT expression in human breast cancer cell lines.

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Syed M Meeran

Full Text Available BACKGROUND: Sulforaphane (SFN, an isothiocyanate found in cruciferous vegetables, is a common dietary component that has histone deacetylase inhibition activity and exciting potential in cancer prevention. The mechanisms by which SFN imparts its chemopreventive properties are of considerable interest and little is known of its preventive potential for breast cancer. PRINCIPAL FINDINGS: We found that SFN significantly inhibits the viability and proliferation of breast cancer cells in vitro while it has negligible effects on normal breast cells. Inhibition of telomerase has received considerable attention because of its high expression in cancer cells and extremely low level of expression in normal cells. SFN treatment dose- and time-dependently inhibited human telomerase reverse transcriptase (hTERT, the catalytic regulatory subunit of telomerase, in both MCF-7 and MDA-MB-231 human breast cancer cells. DNA methyltransferases (DNMTs, especially DNMT1 and DNMT3a, were also decreased in SFN-treated breast cancer cells suggesting that SFN may repress hTERT by impacting epigenetic pathways. Down-regulation of DNMTs in response to SFN induced site-specific CpG demethylation occurring primarily in the first exon of the hTERT gene thereby facilitating CTCF binding associated with hTERT repression. Chromatin immunoprecipitation (ChIP analysis of the hTERT promoter revealed that SFN increased the level of active chromatin markers acetyl-H3, acetyl-H3K9 and acetyl-H4, whereas the trimethyl-H3K9 and trimethyl-H3K27 inactive chromatin markers were decreased in a dose-dependent manner. SFN-induced hyperacetylation facilitated the binding of many hTERT repressor proteins such as MAD1 and CTCF to the hTERT regulatory region. Depletion of CTCF using siRNA reduced the SFN-induced down-regulation of hTERT mRNA transcription in these breast cancer cells. In addition, down-regulation of hTERT expression facilitated the induction of cellular apoptosis in human breast

GSK-3 directly regulates phospho-4EBP1 in renal cell carcinoma cell-line: an intrinsic subcellular mechanism for resistance to mTORC1 inhibition

International Nuclear Information System (INIS)

Ito, Hiromi; Ichiyanagi, Osamu; Naito, Sei; Bilim, Vladimir N.; Tomita, Yoshihiko; Kato, Tomoyuki; Nagaoka, Akira; Tsuchiya, Norihiko

2016-01-01

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 μM. Cell viability, cell cycling and direct interaction between GSK-3β and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3β and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed

DK1 Induces Apoptosis via Mitochondria-Dependent Signaling Pathway in Human Colon Carcinoma Cell Lines In Vitro

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Yazmin Hussin

2018-04-01

Full Text Available Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z-3-hydroxy-1-(2-hydroxyphenyl-3-phenylprop-2-ene-1-one (DK1, was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide(MTT assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G0/G1phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA

Radiation sensitivity of Merkel cell carcinoma cell lines

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Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

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Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

International Nuclear Information System (INIS)

Aravalli, Rajagopal N.; Behnan Sahin, M.; Cressman, Erik N.K.; Steer, Clifford J.

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Establishment and characterization of a unique 1 {mu}m diameter liver-derived progenitor cell line

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Aravalli, Rajagopal N., E-mail: arava001@umn.edu [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Behnan Sahin, M. [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Cressman, Erik N.K. [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 {mu}m in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

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Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

2013-02-15

Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

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Mead, Emma J; Masterton, Rosalyn J; Feary, Marc; Obrezanova, Olga; Zhang, Lin; Young, Robert; Smales, C Mark

2015-12-15

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity. © 2015 Authors; published by Portland Press Limited.

Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva.

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Girjes, Adeeb A; Lee, Kristen E; Carrick, Frank N

2003-01-01

A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal tool for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.

Hypoxic cell turnover in different solid tumor lines

International Nuclear Information System (INIS)

Ljungkvist, Anna S.E.; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

2005-01-01

Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h

Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

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Hung Jaclyn Y

2008-09-01

Full Text Available Abstract Background Musashi1 (Msi1 is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. Methods We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. Results We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Conclusion Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Cytotoxicity of Labruscol, a New Resveratrol Dimer Produced by Grapevine Cell Suspensions, on Human Skin Melanoma Cancer Cell Line HT-144

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Laetitia Nivelle

2017-11-01

Full Text Available A new resveratrol dimer (1 called labruscol, has been purified by centrifugal partition chromatography of a crude ethyl acetate stilbene extract obtained from elicited grapevine cell suspensions of Vitis labrusca L. cultured in a 14-liter stirred bioreactor. One dimensional (1D and two dimensional (2D nuclear magnetic resonance (NMR analyses including 1H, 13C, heteronuclear single-quantum correlation (HSQC, heteronuclear multiple bond correlation (HMBC, and correlation spectroscopy (COSY as well as high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS were used to characterize this compound and to unambiguously identify it as a new stilbene dimer, though its relative stereochemistry remained unsolved. Labruscol was recovered as a pure compound (>93% in sufficient amounts (41 mg to allow assessment of its biological activity (cell viability, cell invasion and apoptotic activity on two different cell lines, including one human skin melanoma cancer cell line HT-144 and a healthy human dermal fibroblast (HDF line. This compound induced almost 100% of cell viability inhibition in the cancer line at a dose of 100 μM within 72 h of treatment. However, at all tested concentrations and treatment times, resveratrol displayed an inhibition of the cancer line viability higher than that of labruscol in the presence of fetal bovine serum. Both compounds also showed differential activities on healthy and cancer cell lines. Finally, labruscol at a concentration of 1.2 μM was shown to reduce cell invasion by 40%, although no similar activity was observed with resveratrol. The cytotoxic activity of this newly-identified dimer is discussed.

Deficient expression of enhanced reactivation of parvovirus H-1 in ataxia telangiectasia cells irradiated with X-rays or u.v. light

International Nuclear Information System (INIS)

Gilgers, Genevieve; Chen, Y.Q.; Cornelis, J.J.; Rommelaere, Jean

1987-01-01

Cells of patients with ataxia telangiectasia (AT), an inherited disease characterized by a high propensity to cancer, are hypersensitive to ionizing radiation. We investigated whether the hyper-radiosensitivity of AT cells correlated with a defect in their constitutive and/or conditional ability to rescue a damaged exogenous virus. For that purpose, parvovirus H-1, a single-stranded DNA virus whose intranuclear replication mostly relies on host cell functions, was used as a probe. The survival of u.v.-or γ-irradiated H-1 was measured in X-, u.v.-or mock-irradiated human cells of normal (NB-E) or AT (AT5BIVA) origin. γ-Irradiated H-1 survived to similar extents in untreated normal and AT cell lines. Both X- and u.v.-irradiation induced normal cells to achieve an enhanced reactivation (ER) of γ- or u.v.-damaged H-1. In contrast, neither dose-effect curves nor time course revealed significant levels of ER expression after X- or u.v.-irradiation in AT5BIVA cells. Our results suggest that the impairment of ER of damaged parvoviruses may constitute a marker of the AT cell phenotype and be related to the radiosensitivity of AT cells. (author)

Deficient expression of enhanced reactivation of parvovirus H-1 in ataxia telangiectasia cells irradiated with X-rays or u. v. light

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Hilgers, G.; Chen, Y.Q.; Cornelis, J.J.; Rommelaere, J.

1987-02-01

Cells of patients with ataxia telangiectasia (AT), an inherited disease characterized by a high propensity to cancer, are hypersensitive to ionizing radiation. We investigated whether the hyper-radiosensitivity of AT cells correlated with a defect in their constitutive and/or conditional ability to rescue a damaged exogenous virus. For that purpose, parvovirus H-1, a single-stranded DNA virus whose intranuclear replication mostly relies on host cell functions, was used as a probe. The survival of u.v.- or gamma-irradiated H-1 was measured in X-, u.v.- or mock-irradiated human cells of normal (NB-E) or AT (AT5BIVA) origin. gamma-Irradiated H-1 survived to similar extents in untreated normal and AT cell lines. Both X- and u.v.-irradiation induced normal cells to achieve an enhanced reactivation (ER) of gamma- or u.v.-damaged H-1. In contrast, neither dose-effect curves nor time course revealed significant levels of ER expression after X- or u.v.-irradiation in AT5BIVA cells. Our results suggest that the impairment of ER of damaged parvoviruses may constitute a marker of the AT cell phenotype and be related to the radiosensitivity of AT cells.

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

Science.gov (United States)

Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

2001-01-01

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

Lithium-Acetate-Mediated Biginelli One-Pot Multicomponent Synthesis under Solvent-Free Conditions and Cytotoxic Activity against the Human Lung Cancer Cell Line A549 and Breast Cancer Cell Line MCF7

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Harshita Sachdeva

2012-01-01

Full Text Available Various Biginelli compounds (dihydropyrimidinones have been synthesized efficiently and in high yields under mild, solvent-free, and eco-friendly conditions in a one-pot reaction of 1,3-dicarbonyl compounds, aldehydes, and urea/thiourea/acetyl thiourea using lithium-acetate as a novel catalyst without the addition of any proton source. Comparative catalytic efficiency of lithium-acetate and polyphosphoric acid to catalyze Biginelli condensation is also studied under neat conditions. The reaction is carried out in the absence of any solvent and represents an improvement of the classical Biginelli protocol and an advantage in comparison with FeCl3·6H2O, NiCl2·6H2O and CoCl2·6H2O that were used with HCl as a cocatalyst. Compared to classical Biginelli reaction conditions, the present method has advantages of good yields, short reaction times, and experimental simplicity. The obtained products have been identified by spectral (1H NMR and IR data and their melting points. The prepared compounds are evaluated for anticancer activity against two human cancer cell lines (lung cancer cell line A549 and breast cancer cell line MCF7.

Regulation of voltage-gated potassium channels attenuates resistance of side-population cells to gefitinib in the human lung cancer cell line NCI-H460.

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Choi, Seon Young; Kim, Hang-Rae; Ryu, Pan Dong; Lee, So Yeong

2017-02-21

Side-population (SP) cells that exclude anti-cancer drugs have been found in various tumor cell lines. Moreover, SP cells have a higher proliferative potential and drug resistance than main population cells (Non-SP cells). Also, several ion channels are responsible for the drug resistance and proliferation of SP cells in cancer. To confirm the expression and function of voltage-gated potassium (Kv) channels of SP cells, these cells, as well as highly expressed ATP-binding cassette (ABC) transporters and stemness genes, were isolated from a gefitinib-resistant human lung adenocarcinoma cell line (NCI-H460), using Hoechst 33342 efflux. In the present study, we found that mRNA expression of Kv channels in SP cells was different compared to Non-SP cells, and the resistance of SP cells to gefitinib was weakened with a combination treatment of gefitinib and Kv channel blockers or a Kv7 opener, compared to single-treatment gefitinib, through inhibition of the Ras-Raf signaling pathway. The findings indicate that Kv channels in SP cells could be new targets for reducing the resistance to gefitinib.

Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1 Improves Recombinant Protein Expression in the HEK293 Cell Line

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Laura Abaandou